Method for producing an ActRIIB-Fc fusion polypeptide

ABSTRACT

In certain aspects, the present invention provides compositions and methods for modulating (promoting or inhibiting) growth of a tissue, such as bone, cartilage, muscle, fat, brown fat and/or neuronal tissue and for treating metabolic disorders such as diabetes and obesity, as well as disorders associated with any of the foregoing tissue.

CROSS-REFERENCE TO RELATED APPLICATIONS:

This application is a continuation of U.S. application Ser. No.14/814,040, filed Jul. 30, 2015, which application is a continuation ofU.S. application Ser. No. 13,657,649, filed Oct. 22, 2012 (now U.S. Pat.No. 9,181,533), which is a divisional of U.S. patent application Ser.No. 12/796,307, filed Jun. 8, 2010 (now U.S. Pat. No. 8,293,881), whichclaims the benefit of U.S. Provisional Application Nos. 61/280,543,filed Nov. 3, 2009, and 61/268,420, filed Jun. 12, 2009. Thespecifications of each of the foregoing applications are incorporatedherein by reference in their entirety.

SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in ASCII format and is hereby incorporated byreference in its entirety. Said ASCII copy, created on Jul. 26, 2017, isnamed 1848179-0002-045-104_SL.txt and is 27,678 bytes in size.

BACKGROUND OF THE INVENTION

The transforming growth factor-beta (TGF-beta) superfamily contains avariety of growth factors that share common sequence elements andstructural motifs. These proteins are known to exert biological effectson a large variety of cell types in both vertebrates and invertebrates.Members of the superfamily perform important functions during embryonicdevelopment in pattern formation and tissue specification and caninfluence a variety of differentiation processes, includingadipogenesis, myogenesis, chondrogenesis, cardiogenesis, hematopoiesis,neurogenesis, and epithelial cell differentiation. The family isrepresented by proteins named, variously, the activins and inhibins,TGF-beta, Growth and Differentiation Factors (GDFs) and BoneMorphogenetic Factors (BMPs). Other members of the family are alsoknown, such as Nodal and Lefty. By manipulating the activity of a memberof the TGF-beta family, it is often possible to cause significantphysiological changes in an organism. For example, the Piedmontese andBelgian Blue cattle breeds carry a loss-of-function mutation in the GDF8(also called myostatin) gene that causes a marked increase in musclemass. Grobet et al., Nat Genet. 1997, 17(1):71-4. Furthermore, inhumans, inactive alleles of GDF8 are associated with increased musclemass and, reportedly, exceptional strength. Schuelke et al., N Engl JMed 2004, 350:2682-8.

Changes in muscle, bone, fat, cartilage and other tissues may beachieved by agonizing or antagonizing signaling that is mediated by anappropriate TGF-beta family member. Thus, there is a need for agentsthat function as potent regulators of signaling by members of theTGF-beta superfamily.

SUMMARY OF THE INVENTION

In certain aspects, the present disclosure provides novel ActRIIBpolypeptides, particularly amino- and carboxy-terminal truncations andsequence alterations. In one embodiment, polypeptides including aminoacids 25-131 of human ActRIIB (SEQ ID NO:1) or variants thereof, aredescribed. Such polypeptides are demonstrated to have surprisingefficacy in the treatment of a variety of disorders, but particularlydisorders associated with obesity, insulin resistance and othermetabolic disorders. ActRIIB polypeptides disclosed herein can be usedto have a variety of desirable effects in patients, including, forexample, increasing lean body mass, decreasing white fat mass,increasing brown fat mass, decreasing serum triglycerides, decreasingserum insulin levels or decreasing serum free fatty acid levels. ActRIIBpolypeptides disclosed herein may be used for the treatment of a varietyof disorders or conditions, including muscle and neuromuscular disorders(e.g., muscular dystrophy, amyotrophic lateral sclerosis (ALS), andmuscle atrophy), adipose tissue disorders (e.g., obesity, fatty liverdisease), metabolic disorders (e.g., type 2 diabetes, insulinresistance, metabolic syndrome), neurodegenerative disorders, and musclewasting associated with old age (sarcopenia), prostate cancer therapy(e.g., androgen deprivation therapy), and cachexia associated with avariety of cancers. Examples of ActRIIB polypeptides include a humanActRIIB-Fc fusion protein set forth in SEQ ID NO:8 and described hereinas ActRIIB(25-131)-hFc.

In certain aspects, the disclosure provides novel polypeptides that arederived from ActRIIB (referred to as ActRIIB polypeptides). In someembodiments, a polypeptide may be selected from the group consisting of:a polypeptide comprising an amino acid sequence wherein the amino acidsequence consists of the sequence of SEQ ID NO:8 or an amino acidsequence that differs from SEQ ID NO:8 at no more than one, two, three,four or five amino acid positions; a polypeptide produced by theexpression in a mammalian cell of the nucleic acid of SEQ ID NO: 4 or anucleic acid that hybridizes under stringent condition to the complementthereof; a polypeptide produced by the expression in a mammalian cell ofthe nucleic acid of SEQ ID NO:6 or a nucleic acid that hybridizes understringent conditions to the complement thereof. A polypeptide disclosedherein may comprise a portion derived from ActRIIB and one or moreheterologous portions, wherein the portion derived from ActRIIB maycomprise an amino acid sequence consisting of the sequence of aminoacids 25-131 of SEQ ID NO:1 or an amino acid sequence that differs thesequence of amino acids 25-131 of SEQ ID NO:1 at no more than one, two,three, four or five amino acid positions. The heterologous portion maycomprise a constant domain of an immunoglobulin, an Fc domain of animmunoglobulin or, particularly, an Fc domain of a human IgG1 (the term“human IgG1 shall be understood to include variants of such Fc that arecompatible with use in humans). ActRIIB polypeptides may include aportion derived from ActRIIB that comprises an amino acid sequenceconsisting of the sequence of amino acids 25-131 of SEQ ID NO:1. AnActRIIB polypeptide disclosed herein may be such that the amino terminushas the sequence ETR. An ActRIIB polypeptide disclosed herein may causea statistically significant increase in lean body mass in a mouse afterfour weeks of treatment twice per week at a dose level of 10 mg/kg. Themean increase of lean tissue mass may be at least 1, 2, 3, 4 or 5 ormore grams. An ActRIIB polypeptide disclosed herein may cause astatistically significant decrease in fat mass in a mouse fed a high fatdiet after four weeks of treatment twice per week at a dose level of 10mg/kg. The mean decrease in fat mass may be 5, 7, 10, 15 or more grams.An ActRIIB polypeptide disclosed herein may cause a statisticallysignificant decrease in serum triglyceride levels in a mouse fed a highfat diet after four weeks of treatment twice per week at a dose level of10 mg/kg. The mean decrease in serum triglycerides may be at least 50,75, 100, 125 or 150 or more mg/dl. An ActRIIB polypeptide disclosedherein may cause a statistically significant decrease in serum freefatty acid levels in a mouse fed a high fat diet after four weeks oftreatment twice per week at a dose level of 10 mg/kg. The mean decreasein free fatty acids may be at least 500, 750, 1000 or more micromoles/dlfree fatty acids. An ActRIIB polypeptide disclosed herein may cause astatistically significant decrease in serum insulin levels in a mousefed a high fat diet after four weeks of treatment twice per week at adose level of 10 mg/kg. The mean decrease in serum insulin may be atleast 0.5, 1, 1.5, 2 or more ng/ml insulin. As used herein, the term“statistically significant” generally refers to a p value or >0.05, butother measures of significance may be recognized for different types ofstatistical tests, and in such cases, the term “statisticallysignificant” should use the most widely used formula for assessing thesignificance of the data. ActRIIB polypeptides may comprise at least oneN-linked sugar, and may include two, three or more N-linked sugars. Suchpolypeptides may also comprise O-linked sugars. ActRIIB polypeptides maybe produced in a variety of cell lines that glycosylate the protein in amanner that is suitable for patient use, including engineered insect oryeast cells, and mammalian cells such as COS cells, CHO cells, HEK cellsand NSO cells. ActRIIB polypeptides may form covalent or non-covalentdimers, including homodimers. Generally. Fc fusion proteins tend to formhomodimers that are covalently linked. Any of the foregoing polypeptidesmay be incorporated into a pharmaceutical preparation.

In certain aspects, the ActRIIB polypeptides disclosed herein bind to anActRIIB ligand such as GDF8, GDF11, activin, BMP7, GDF3 or nodal.Optionally, an ActRIIB polypeptide binds to an ActRIIB ligand with a Kdless than 10 micromolar or less than 1 micromolar, 100, 10, 1 or 0.1nanomolar. An ActRIIB polypeptide disclosed herein may include one, two,three, four, five or more alterations in the amino acid sequence (e.g.,in the ligand-binding domain) relative to a naturally occurring ActRIIBpolypeptide. The alteration in the amino acid sequence may, for example,alter glycosylation of the polypeptide when produced in a mammalian,insect or other eukaryotic cell or alter proteolytic cleavage of thepolypeptide relative to the naturally occurring ActRIIB polypeptide. AnActRIIB polypeptide may be a fusion protein that has, as one domain, anamino acid sequence derived from ActRIIB (e.g., a ligand-binding domainof an ActRIIB or a variant thereof) and one or more additional domainsthat provide a desirable property, such as improved pharmacokinetics,easier purification, targeting to particular tissues, etc. For example,a domain of a fusion protein may enhance one or more of in vivostability, in vivo half life, uptake/administration, tissue localizationor distribution, formation of protein complexes, multimerization of thefusion protein, and/or purification. An ActRIIB fusion protein mayinclude an immunoglobulin Fc domain (wild-type or mutant) or a serumalbumin. In certain embodiments, an ActRIIB-Fc fusion comprises arelatively unstructured linker positioned between the Fc domain and theextracellular ActRIIB domain. This unstructured linker may correspond tothe roughly 15 amino acid unstructured region at the C-terminal end ofthe extracellular domain of ActRIIB (the “tail”), or it may be anartificial sequence of between 5 and 15, 20, 30, 50 or more amino acidsthat are relatively free of secondary structure. A linker may be rich inglycine and proline residues and may, for example, contain repeatingsequences of threonine/serine and glycines (e.g., TG₄ or SG₄ repeats).In the context of a polypeptide of SEQ ID NO:8, it appears to beadvantageous to use a short, flexible linker, such as one, two, three,four or five glycine residues, optionally with one or more smallresidues such as alanine, threonine or serine. A fusion protein mayinclude a purification subsequence, such as an epitope tag, a FLAG tag,a polyhistidine sequence, and a GST fusion. Optionally, an ActRIIBpolypeptide includes one or more modified amino acid residues selectedfrom: a glycosylated amino acid, a PEGylated amino acid, a farnesylatedamino acid, an acetylated amino acid, a biotinylated amino acid, anamino acid conjugated to a lipid moiety, and an amino acid conjugated toan organic derivatizing agent.

In certain aspects, an ActRIIB polypeptide may be formulated as apharmaceutical preparation. A pharmaceutical preparation will preferablybe pyrogen free (meaning pyrogen free to the extent required byregulations governing the quality of products for therapeutic use). Apharmaceutical preparation may also include one or more additionalcompounds such as a compound that is used to treat an ActRIIB-associateddisorder.

In certain aspects, the disclosure provides nucleic acids encoding anActRIIB polypeptide. Such a nucleic acid may comprises a nucleic acidsequence of 73-396 of SEQ ID NO:4 or one that hybridizes under stringentconditions to the complement of nucleotides 73-396 of SEQ ID NO:4. Anucleic acid may one that comprises the sequence of SEQ ID NO:4. Such anucleic acid may comprises a nucleic acid sequence of 73-396 of SEQ IDNO:6 or one that hybridizes under stringent conditions to the complementof nucleotides 73-396 of SEQ ID NO:6. A nucleic acid may one thatcomprises the sequence of SEQ ID NO:6. In certain aspects, an ActRIIBprotein may be expressed in a mammalian cell line that mediates suitablynatural glycosylation of the ActRIIB protein so as to diminish thelikelihood of an unfavorable immune response in a patient (including thepossibility of veterinary patients). Human and CHO cell lines have beenused successfully, and it is expected that other common mammalianexpression vectors will be useful. Thus the disclosure provides culturedcells comprising any of the nucleic acids disclosed herein. Such cellsmay be mammalian cells, including CHO cells, NSO cells, HEK cells andCOS cells. Other cells may be chosen depending on the species of theintended patient. Other cells are disclosed herein. Cultured cells areunderstood to mean cells maintained in laboratory or other man-madeconditions (e.g., frozen, or in media) and not part of a livingorganism.

In certain aspects, the disclosure provides methods for making a ActRIIBpolypeptide. Such a method may include expressing any of the nucleicacids (e.g., SEQ ID NO: 4 or 6, and nucleic acids that hybridize theretounder stringent conditions) disclosed herein in a suitable cell, such asa Chinese hamster ovary (CHO) cell. Such a method may comprise: a)culturing a cell under conditions suitable for expression of the ActRIIBpolypeptide, wherein said cell is transformed with an ActRIIB expressionconstruct; and b) recovering the ActRIIB polypeptide so expressed.ActRIIB polypeptides may be recovered as crude, partially purified orhighly purified fractions using any of the well known techniques forobtaining protein from cell cultures as well as techniques describedherein.

In certain aspects the disclosure provides methods for treating asubject having a disorder associated with muscle loss or insufficientmuscle growth. Such a method may comprise administering to the subjectan effective amount of any of the foregoing ActRIIB polypeptides orpharmaceutical preparations thereof.

In certain aspects the disclosure provides methods for increasing thelean mass or reducing the rate of loss of lean mass in a subject in needthereof. Such a method may comprise administering to the subject aneffective amount of any of the foregoing ActRIIB polypeptides orpharmaceutical preparations thereof.

In certain aspects, the disclosure provides methods for decreasing thebody fat content or reducing the rate of increase in body fat content ina subject. Such a method may comprise administering to the subject aneffective amount of any of the foregoing ActRIIB polypeptides orpharmaceutical preparations thereof.

In certain aspects, the disclosure provides methods for treating adisorder associated with undesirable body weight gain in a subject. Sucha method may comprise administering to the subject an effective amountof any of the foregoing ActRIIB polypeptides or pharmaceuticalpreparations thereof.

In certain aspects, the disclosure provides methods for treating ametabolic disorder in a subject. Such a method may compriseadministering to the subject an effective amount of any of the foregoingActRIIB polypeptides or pharmaceutical preparations thereof. A patienteligible for treatment may have one or more of the followingcharacteristics: elevated serum triglyceride levels; elevated free fattyacid levels; or elevated serum insulin levels. Examples of metabolicdisorders include type 2 diabetes, metabolic syndrome, insulinresistance and obesity.

In certain aspects, an ActRIIB polypeptide disclosed herein may be usedin a method for treating a subject having a disorder associated withmuscle loss or insufficient muscle growth. Such disorders include muscleatrophy, muscular dystrophy, amyotrophic lateral sclerosis (ALS), and amuscle wasting disorder (e.g., cachexia, anorexia, DMD syndrome, BMDsyndrome, AIDS wasting syndrome, muscular dystrophies, neuromusculardiseases, motor neuron diseases, diseases of the neuromuscular junction,and inflammatory myopathies). A method may comprise administering to asubject in need thereof an effective amount of an ActRIIB polypeptide.

In certain aspects, an ActRIIB polypeptide disclosed herein may be usedin a method for decreasing the body fat content or reducing the rate ofincrease in body fat content, and for treating a disorder associatedwith undesirable body weight gain, such as obesity, non-insulindependent diabetes mellitus (NIDDM), cardiovascular disease, cancer,hypertension, osteoarthritis, stroke, respiratory problems, and gallbladder disease. These methods may comprise administering to a subjectin need thereof an effective amount of an ActRIIB polypeptide.

In certain specific aspects, an ActRIIB polypeptide disclosed herein maybe used in a method for treating a disorder associated with abnormalactivity of GDF8. Such disorders include metabolic disorders such astype 2 diabetes, impaired glucose tolerance, metabolic syndrome (e.g.,syndrome X), and insulin resistance induced by trauma (e.g., burns ornitrogen imbalance); adipose tissue disorders (e.g., obesity); musculardystrophy (including Duchenne muscular dystrophy); amyotrophic lateralsclerosis (ALS); muscle atrophy; organ atrophy; frailty; carpal tunnelsyndrome; congestive obstructive pulmonary disease; sarcopenia, cachexiaand other muscle wasting syndromes; osteoporosis; glucocorticoid-inducedosteoporosis; osteopenia; osteoarthritis; osteoporosis-relatedfractures; low bone mass due to chronic glucocorticoid therapy,premature gonadal failure, androgen suppression, vitamin D deficiency,secondary hyperparathyroidism, nutritional deficiencies, and anorexianervosa. The method may comprise administering to a subject in needthereof an effective amount of an ActRIIB polypeptide.

In certain aspects, the disclosure provides a method for identifying anagent that stimulates growth of a tissue such as bone, cartilage, muscleand fat. The method comprises: a) identifying a test agent that binds toa ligand-binding domain of an ActRIIB polypeptide competitively with anActRIIB polypeptide; and b) evaluating the effect of the agent on growthof the tissue.

In certain aspects, the disclosure provides methods for antagonizingactivity of an ActRIIB polypeptide or an ActRIIB ligand (e.g., GDF8,GDF11, activin, GDF3, BMP7, and Nodal) in a cell. The methods comprisecontacting the cell with an ActRIIB polypeptide. Optionally, theactivity of the ActRIIB polypeptide or the ActRIIB ligand is monitoredby a signaling transduction mediated by the ActRIIB/ActRIIB ligandcomplex, for example, by monitoring cell proliferation. The cells of themethods include an osteoblast, a chondrocyte, a myocyte, an adipocyteand a muscle cell.

In certain aspects, the disclosure provides uses of an ActRIIBpolypeptide for making a medicament for the treatment of a disorder orcondition as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

The patent or application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Patent Office upon request andpayment of the necessary fee.

FIG. 1 shows the full, unprocessed amino acid sequence forActRIIB(25-131)-hFc (SEQ ID NO:3). The TPA leader (residues 1-22) anddouble-truncated ActRIIB extracellular domain (residues 24-131, usingnumbering based on the native sequence in SEQ ID NO: 1) are eachunderlined. Highlighted is the glutamate revealed by sequencing to bethe N-terminal amino acid of the mature fusion protein, which is atposition 25 relative to SEQ ID NO:1.

FIGS. 2A and 2B show a nucleotide sequence encoding ActRIIB(25-131)-hFc(the coding strand is shown at top, SEQ ID NO:4, and the complementshown at bottom 3′-5′, SEQ ID NO:5). Sequences encoding the TPA leader(nucleotides 1-66) and ActRIIB extracellular domain (nucleotides 73-396)are underlined. The corresponding amino acid sequence forActRIIB(25-131) is also shown.

FIGS. 3A and 3B show an alternative nucleotide sequence encodingActRIIB(25-131)-hFc (the coding strand is shown at top, SEQ ID NO:6, andthe complement shown at bottom 3′-5′, SEQ ID NO:7). This sequenceconfers a greater level of protein expression in initial transformants,making cell line development a more rapid process. Sequences encodingthe TPA leader (nucleotides 1-66) and ActRIIB extracellular domain(nucleotides 73-396) are underlined, and substitutions in the wild typenucleotide sequence of the ECD (see FIGS. 2A and 2B) are highlighted.The corresponding amino acid sequence for ActRIIB(25-131) is also shown.

FIG. 4 shows the effect of four weeks treatment with ActRIIB(25-131)-hFcon lean tissue mass in mouse. Vehicle was Tris-buffered saline (TBS).Data are means (n=10 per group)±SEM. **, P<0.01 vs. TBS by unpairedt-test. ActRIIB(25-131)-hFc treatment increased lean tissue mass in aclear dose-dependent manner.

FIG. 5 shows the effect of four weeks treatment with ActRIIB(25-131)-hFcon pectoralis muscle mass in mouse. Vehicle was Tris-buffered saline(TBS). Data are means (n=10 per group)±SEM. *, P<0.05; **, P<0.01 vs.TBS by unpaired t-test. ActRIIB(25-131)-hFc treatment increasedpectoralis muscle mass in a clear dose-dependent manner.

FIG. 6 shows the effect of ActRIIB(25-131)-hFc treatment on gripstrength in mouse. Vehicle was Tris-buffered saline (TBS). Data aremeans (n=10 per group). **, P<0.01 vs. TBS by unpaired t-test.ActRIIB(25-131)-hFc treatment increased grip strength in adose-dependent manner.

FIG. 7 shows the effect of four weeks treatment with ActRIIB(25-131)-hFcon lean tissue mass in a mouse model of androgen deprivation. Vehiclewas Tris-buffered saline (TBS). Data for orchidectomized (ORX) orsham-operated mice are means (n=10 per group)±SD. ***, P<0.001 vs. TBScontrol. ActRIIB(25-131)-hFc increased lean tissue mass as effectivelyas did its full-length counterpart ActRIIB(20-134)-mFc.

FIG. 8 shows the effect of ActRIIB(25-131)-hFc on lean tissue mass in amouse model of diet-induced obesity. Vehicle was Tris-buffered saline(TBS). Data are means (n=9-10 per group). ***, P<0.001 vs. TBS control.ActRIIB(25-131)-hFc increased lean tissue mass effectively in mice fed ahigh fat diet.

FIG. 9 shows the effect of ActRIIB(25-131)-hFc on fat mass in a mousemodel of diet-induced obesity. Vehicle was Tris-buffered saline (TBS).Data are means (n=9-10 per group)±SD. *, P<0.05; ***, P<0.001 vs. TBScontrol. Compared to vehicle, ActRIIB(25-131)-hFc treatment for 12 weeksreduced fat mass by approximately half in mice fed a high fat diet.

FIG. 10 depicts serum triglyceride concentrations in mice as a functionof diet and ActRIIB(25-131)-hFc treatment for 60 days. Data aremeans±SEM. ***, P<0.001. In mice fed a high-fat diet,ActRIIB(25-131)-hFc reduced triglyceride concentrations by more than50%, thereby normalizing triglycerides to levels observed instandard-diet controls.

FIG. 11 depicts serum free fatty acid (FFA) concentrations in mice as afunction of diet and ActRIIB(25-131)-hFc treatment for 60 days. Data aremeans±SEM. ***, P<0.001. In mice fed a high-fat diet,ActRIIB(25-131)-hFc reduced FFA concentrations by nearly 55%, therebynormalizing FFA to levels observed in standard-diet controls.

FIG. 12 depicts serum high-density lipoprotein (HDL) concentrations inmice as a function of diet and ActRIIB(25-131)-hFc treatment for 60days. Data are means±SEM. ***, P<0.001. In mice fed a high-fat diet,ActRIIB(25-131)-hFc reduced HDL concentrations by nearly 50%, therebynormalizing HDL to levels observed in standard-diet controls.

FIG. 13 depicts serum low-density lipoprotein (LDL) concentrations inmice as a function of diet and ActRIIB(25-131)-hFc treatment for 60days. Data are means±SEM. *, P<0.05. In mice fed a high-fat diet,ActRIIB(25-131)-hFc reduced LDL concentrations by more than 40%.

FIG. 14 depicts serum insulin concentrations in mice as a function ofdiet and ActRIIB(25-131)-hFc treatment for 60 days. Data are means±SEM.**, P<0.01. In mice fed a high-fat diet, ActRIIB(25-131)-hFc reducedinsulin concentrations by more than 60%, thereby normalizing insulin tolevels observed in standard-diet controls.

FIG. 15 depicts serum adiponectin concentrations in mice as a functionof diet and ActRIIB(25-131)-hFc treatment for 60 days. ELISAmeasurements detect all main oligomeric isoforms (total adiponectin),and data are means±SEM. **, P<0.01; ***, P<0.001. In mice fed a high-fatdiet, ActRIIB(25-131)-hFc increased adiponectin concentrations by morethan 75% and even boosted adiponectin significantly above the levelsobserved in standard-diet controls.

FIG. 16 shows thermogenic histological changes induced within epididymalwhite adipose tissue by ActRIIB(25-131)-hFc treatment for 60 days in amouse model of diet-induced obesity. All microscopic images shown at thesame magnification. Hematoxylin and eosin (H&E) staining indicates theability of ActRIIB(25-131)-hFc to reduce lipid droplet size and induceclusters of multilocular adipocytes (arrows) characteristic of brownfat. Immunostaining of non-adjacent sections reveals widespreadcytoplasmic induction of UCP1 (green fluorescence) in both multilocularand unilocular adipocytes.

FIG. 17 shows the effect of ActRIIB(25-131)-hFc treatment for 60 days onUCP1 mRNA levels in epididymal white fat in a mouse model ofdiet-induced obesity. Data obtained by reverse transcriptase polymerasechain reaction (RT-PCR), in relative units (RU), are means±SEM; n=6-7per group; *, p<0.05. ActRIIB(25-131)-hFc caused a 60-fold increase inmRNA encoding this selective marker for brown fat, thus indicatingupregulation of thermogenic capability within this white fat depot.

FIG. 18 shows levels of adiponectin mRNA in epididymal white fat of miceas a function of diet and ActRIIB(25-131)-hFc treatment for 60 days.RT-PCR data, in relative units (RU), are means±SEM; n=7 per group; *,p<0.05. In mice fed a high-fat diet, ActRIIB(25-131)-hFc increasedadiponectin mRNA levels by more than 60%, thus contributing to elevatedconcentrations of circulating adiponectin in these mice.

FIG. 19 shows the effect of ActRIIB(25-131)-hFc treatment for 60 days onfatty liver deposits (hepatic steatosis) in a mouse model ofdiet-induced obesity. Liver sections (all shown at the samemagnification) stained with Oil Red O reveal pronounced lipid depositionunder high-fat dietary conditions but not control conditions. Arrowsindicate several of many densely packed lipid droplets, which arestained bright red but difficult to discern in black-and-white images.ActRIIB(25-131)-hFc inhibited formation of such lipid droplets andlargely restored the appearance of liver tissue to that of mice fed thestandard diet.

FIG. 20 shows the effect of ActRIIB(25-131)-mFc treatment for 35 days onthe distribution of abdominal fat in a mouse model of diet-inducedobesity. Visceral and subcutaneous fat depots were detected anddifferentiated in vivo by micro-computed tomography (microCT)encompassing spinal cord segments T13-L5. N=4 per group; scale bar=5 mm.Compared to controls fed a high-fat diet, ActRIIB(25-131)-mFc treatmentreduced the volume of both visceral and subcutaneous depots of abdominalfat.

FIG. 21 shows the effect of ActRIIB(25-131)-mFc treatment for 60 days onthe volume of visceral fat as determined by microCT in a mouse model ofdiet-induced obesity. Data are means±SEM; n=4 per group; ***, P<0.001.In mice fed a high-fat diet. ActRIIB(25-131)-mFc reduced the volume ofvisceral fat by more than 60% compared to vehicle.

FIG. 22 shows the effect of ActRIIB(25-131)-mFc treatment for 60 days onthe volume of abdominal subcutaneous fat as determined by microCT in amouse model of diet-induced obesity. Data are means±SEM; n=4 per group;***, P<0.001. In mice fed a high-fat diet, ActRIIB(25-131)-mFc reducedthe volume of subcutaneous fat by nearly 60% compared to vehicle.

FIG. 23 shows photographs of bilateral pairs of interscapular brown fatdepots as a function of diet and ActRIIB(25-131)-mFc treatment for 60days in a mouse model of diet-induced obesity. High-fat diet increasedthe size and lightened the color of the depots, whereasActRIIB(25-131)-mFc largely reversed these changes.

FIG. 24 depicts the effect of ActRIIB(25-131)-mFc treatment for 60 dayson the mass of interscapular brown fat in a mouse model of diet-inducedobesity. Data are means±SEM for combined left and right depots; ***,p<0.001. ActRIIB(25-131)-mFc reversed the effect of high-fat diet on themass of this brown fat depot.

FIG. 25 depicts the effect of ActRIIB(25-131)-mFc treatment for 60 dayson the density of interscapular brown fat as determined by microCT in amouse model of diet-induced obesity. Data (means±SEM) are expressed instandardized units based on a positive value for the bone mineralhydroxyapatite (HA) and a value of zero for water; therefore, fat valuesare negative, with values for white fat typically close to −120. **,p<0.01. ActRIIB(25-131)-mFc completely reversed the effect of high-fatdiet on the density of this brown fat depot.

FIG. 26 depicts the effect of ActRIIB(25-131)-mFc treatment on leantissue mass as determined in a mouse model of aging by nuclear magneticresonance (NMR) analysis at multiple time points. Data are means of10-15 mice per group per time point; ***, P<0.001 vs. vehicle at sametime point. After 7 weeks of dosing, lean tissue mass in aged micetreated with ActRIIB(25-131)-mFc increased nearly 20% from baseline, incontrast to essentially unchanged values in vehicle-treated controls.

FIG. 27 depicts the effect of ActRIIB(25-131)-mFc treatment on forelimbgrip strength as determined at multiple time points in a mouse model ofaging. Data are means of 13-15 mice per group per time point; **, P<0.01vs. vehicle at same time point. Mice treated with ActRIIB(25-131)-mFcdisplayed an overall trend of increasing grip strength across the study,in contrast to the decline in grip strength observed in vehicle controlsover the same interval.

FIG. 28 depicts the effect of ActRIIB(25-131)-mFc treatment for 8 weekson bone mineral density as determined in a mouse model of aging by dualenergy x-ray absorptiometry (DEXA). Data are means±SEM; *, P<0.05. Bonemineral density in aged mice treated with ActRIIB(25-131)-mFc (n=10)increased significantly compared to vehicle-treated controls (n=14).

FIG. 29 depicts the effect of ActRIIB(25-131)-mFc treatment onwhole-body fat mass as determined in a mouse model of aging by NMRanalysis at multiple time points. Data are means of 10-15 mice per groupper time point. ***, P<0.001 vs. vehicle at same time point. After 7weeks of dosing, fat mass in aged mice treated with ActRIIB(25-131)-mFcexhibited a percent decrease from baseline more than twice the magnitudeof that in vehicle-treated controls.

FIG. 30 depicts the effect of ActRIIB(25-131)-mFc treatment for 8 weekson serum insulin concentrations in a mouse model of aging. Data aremeans±SEM; *, P<0.05. Insulin concentrations in aged mice treated withActRIIB(25-131)-mFc (n=10) were reduced by more than 40% compared tovehicle-treated controls (n=14).

FIG. 31 depicts the effect of ActRIIB(25-131)-mFc treatment for 8 weekson circulating glycated hemoglobin (A C) concentrations. Data aremeans±SEM; n=5-6 per group; **, P<0.01. ActRIIB(25-131)-mFcsignificantly reduced concentrations of glycated hemoglobin, a widelyaccepted indicator of average blood glucose concentrations over anextended period.

FIG. 32 depicts the effect of ActRIIB(25-131)-hFc treatment for 5 weekson lean tissue mass as determined by NMR analysis in a mouse model ofcancer cachexia. Data are means±SEM; ***, P<0.001. In tumor-implantedmice, vehicle treatment (n=7) was associated with a 7% loss in leantissue mass, whereas ActRIIB(25-131)-hFc treatment (n=12) caused a 27%gain in lean tissue mass from baseline.

DETAILED DESCRIPTION

1. Overview

In certain aspects, the present disclosure relates to ActRIIBpolypeptides. As used herein, the term “ActRIIB” refers to a family ofactivin receptor type IIB (ActRIIB) proteins and ActRIIB-relatedproteins, derived from any species. Members of the ActRIIB family aregenerally all transmembrane proteins, composed of a ligand-bindingextracellular domain with cysteine-rich region, a transmembrane domain,and a cytoplasmic domain with predicted serine/threonine kinasespecificity.

The term “ActRIIB polypeptide” is used to refer to polypeptidescomprising any naturally occurring polypeptide of an ActRIIB familymember as well as any variants thereof (including mutants, fragments,fusions, and peptidomimetic forms) that retain a useful activity. Forexample, ActRIIB polypeptides include polypeptides derived from thesequence of any known ActRIIB having a sequence at least about 80%identical to the sequence of an ActRIIB polypeptide, and preferably atleast 85%, 90%, 95%, 97%, 99% or greater identity.

The human ActRIIB precursor has the following amino acid sequence, withthe signal peptide underlined, the extracellular domain indicated inbold, and the potential N-linked glycosylation sites boxed (SEQ IDNO: 1) (NM_001106, 512 aa).

AGGPEVTYEPPPTAPTLLTVLAYSLLPIGGLSLIVLLAFWMYRHRKPPYGHVDIHEDPGPPPPSPLVGLKPLQLLEIKARGRFGCVWKAQLMNDFVAVKIFPLQDKQSWQSEREIFSTPGMKHENLLQFIAAEKRGSNLEVELWLITAFHDKGSLTDYLKGNIITWNELCHVAETMSRGLSYLHEDVPWCRGEGHKPSIAHRDFKSKNVLLKSDLTAVLADFGLAVRFEPGKPPGDTHGQVGTRRYMAPEVLEGAINFQRDAFLRIDMYAMGLVLWELVSRCKAADGPVDEYMLPFEEEICQHPSLEELQEVVVHKKMRPTIKDHWLKHPCLAQLCVTIEECWDHDAEARLSACCVEERVSLIRRSVNGTTSDCLVSLVTSVTNVDLPPKESSI

ActRIIB polypeptides may include any naturally occurring extracellulardomain of an ActRIIB protein as well as any variants thereof (includingmutants, fragments and peptidomimetic forms) that retain a usefulactivity. For example, the extracellular domain of an ActRIIB proteinbinds to a ligand and is generally soluble. The signal sequence can be anative signal sequence of an ActRIIB, or a signal sequence from anotherprotein, such as a tissue plasminogen activator (TPA) signal sequence ora honey bee melatin (HBM) signal sequence.

In part the disclosure provides a novel ActRIIB polypeptide that istruncated, such that the portion derived from ActRIIB is from aminoacids 25-131 of SEQ ID NO:1. As shown herein, polypeptides of this typewhen administered as an Fc construct, ActRIIB(25-131)-hFc, promote theformation of lean body mass (primarily muscle) and the loss of fat mass,while also having marked desirable effects on metabolic parameters suchas serum triglycerides, serum free fatty acids and serum insulin levels.Remarkably, ActRIIB(25-131)-hFc has a much greater effect on thesemetabolic parameters than does a related protein. ActRIIB(20-134). Thesedata are presented in the Examples below.

TGF-β signals are mediated by heteromeric complexes of type I and typeII serine/threonine kinase receptors, which phosphorylate and activatedownstream Smad proteins upon ligand stimulation (Massagué, 2000, NatRev. Mol. Cell Biol. 1:169-178). These type I and type II receptors areall transmembrane proteins, composed of a ligand-binding extracellulardomain with cysteine-rich region, a transmembrane domain, and acytoplasmic domain with predicted serine/threonine specificity. Type Ireceptors are essential for signaling; and type II receptors arerequired for binding ligands and for expression of type I receptors.Type I and II activin receptors form a stable complex after ligandbinding, resulting in phosphorylation of type I receptors by type IIreceptors.

Two related type II receptors, ActRIIA and ActRIIB, have been identifiedas the type II receptors for activins (Mathews and Vale, 1991, Cell65:973-982; Attisano et al., 1992, Cell 68: 97-108). Besides activins,ActRIIA and ActRIIB can biochemically interact with several other TGF-βfamily proteins, including BMP7, Nodal, GDF8, and GDF11 (Yamasbita etal., 1995, J. Cell Biol. 130:217-226; Lee and McPherron, 2001, Proc.Natl. Acad. Sci. 98:9306-9311; Yeo and Whitman, 2001, Mol. Cell 7:949-957; Oh et al., 2002, Genes Dev. 16:2749-54).

In certain embodiments, the present invention relates to antagonizing aligand of ActRIIB receptors (also referred to as an ActRIIB ligand) witha subject ActRIIB polypeptide (e.g., an ActRIIB-Fc polypeptide). Thus,compositions and methods of the present invention are useful fortreating disorders associated with abnormal activity of one or moreligands of ActRIIB receptors. Exemplary ligands of ActRIIB receptorsinclude some TGF-β family members, such as activin, Nodal, GDF3, GDF8,GDF11, and BMP7.

Activins are dimeric polypeptide growth factors and belong to theTGF-beta superfamily. There are three activins (A, B, and AB) that arehomo/heterodimers of two closely related β subunits (β_(A)β_(A),β_(B)β_(B), and β_(A)β_(B)). In the TGF-beta superfamily, activins areunique and multifunctional factors that can stimulate hormone productionin ovarian and placental cells, support neuronal cell survival,influence cell-cycle progress positively or negatively depending on celltype, and induce mesodermal differentiation at least in amphibianembryos (DePaolo et al., 1991, Proc SocEp Biol Med. 198:500-512; Dysonet al., 1997, Curr Biol. 7:81-84; Woodruff, 1998, Biochem Pharmacol.55:953-963). Moreover, erythroid differentiation factor (EDF) isolatedfrom the stimulated human monocytic leukemic cells was found to beidentical to activin A (Murata et al., 1988, PNAS, 85:2434). It wassuggested that activin A acts as a natural regulator of erythropoiesisin the bone marrow. In several tissues, activin signaling is antagonizedby its related heterodimer, inhibin. For example, during the release offollicle-stimulating hormone (FSH) from the pituitary, activin promotesFSH secretion and synthesis, while inhibin prevents FSH secretion andsynthesis. Other proteins that may regulate activin bioactivity and/orbind to activin include follistatin (FS), follistatin-related protein(FSRP), α₂-macroglobulin, Cerberus, and endoglin, which are describedbelow.

Bone morphogenetic protein 7 (BMP7), also called osteogenic protein-1(OP-1), is well known to induce cartilage and bone formation. Inaddition, BMP7 regulates a wide army of physiological processes.Notably, BMP7 has recently been identified as a key promoter of brownadipocyte differentiation (Tseng et al., 2008, Nature 454:1000-1004). Inthis study, genetic ablation of BMP7 led to scarcity of brown fat andnearly complete absence of UCP1 in murine embryos. Moreover,upregulation of BMP7 expression in mice by adenovirus administrationincreased brown fat mass and energy expenditure. Like activin, BMP7binds to type II receptors, ActRIIA and ActRIIB. However, BMP7 andactivin recruit distinct type I receptors into heteromeric receptorcomplexes. The major BMP7 type I receptor observed was ALK2, whileactivin bound exclusively to ALK4 (ActRIIB). BMP7 and activin eliciteddistinct biological responses and activated different Smad pathways(Macias-Silva et al., 1998, J Biol Chem. 273:25628-36).

Growth-and-Differentiation Factor-3 (GDF3), also known as Vg1-related 2,plays an important role in embryonic development and has also beenimplicated in adipogenesis during adulthood. In brief, expression ofGDF3 in white adipose tissue is correlated with body mass or obesity(Weisberg et al., 2003, J Clin Invest 112:1796-1808), andadenovirus-mediated overexpression of GDF3 exaggerates the increase inadiposity observed under high-fat dietary conditions in wildtype mice(Wang et al., 2004, Biochem Biophys Res Conmm 321:1024-1031).Importantly, mice with genetic ablation of GDF3 are healthy andessentially normal when maintained on a standard diet but are protectedfrom obesity, and display an increased basal metabolic rate, whenmaintained on a high-fat diet (Shen et al., 2009, Mol Endocrinol23:113-123). Taken together, these findings implicate GDF3 specificallyin diet-induced obesity and more generally in the regulation ofadiposity.

Nodal proteins have functions in mesoderm and endoderm induction andformation, as well as subsequent organization of axial structures suchas heart and stomach in early embryogenesis. It has been demonstratedthat dorsal tissue in a developing vertebrate embryo contributespredominantly to the axial structures of the notochord and pre-chordalplate while it recruits surrounding cells to form non-axial embryonicstructures. Nodal appears to signal through both type I and type IIreceptors and intracellular effectors known as Smad proteins. Recentstudies support the idea that ActRIIA and ActRIIB serve as type IIreceptors for Nodal (Sakuma et al., Genes Cells. 2002, 7:401-12). It issuggested that Nodal ligands interact with their co-factors (e.g.,cripto) to activate activin type I and type II receptors, whichphosphorylate Smad2. Nodal proteins are implicated in many eventscritical to the early vertebrate embryo, including mesoderm formation,anterior patterning, and left-right axis specification. Experimentalevidence has demonstrated that Nodal signaling activates pAR3-Lux, aluciferase reporter previously shown to respond specifically to activinand TGF-beta. However, Nodal is unable to induce pTlx2-Lux, a reporterspecifically responsive to bone morphogenetic proteins. Recent resultsprovide direct biochemical evidence that Nodal signaling is mediated byboth activin-TGF-beta pathway Smads, Smad2 and Smad3. Further evidencehas shown that the extracellular cripto protein is required for Nodalsignaling, making it distinct from activin or TGF-beta signaling.

Growth and Differentiation Factor-8 (GDF8) is also known as myostatin.GDF8 is a negative regulator of skeletal muscle mass. GDF8 is highlyexpressed in the developing and adult skeletal muscle. The GDF8 nullmutation in transgenic mice is characterized by a marked hypertrophy andhyperplasia of the skeletal muscle (McPherron et al., Nature, 1997,387:83-90). Similar increases in skeletal muscle mass are evident innaturally occurring mutations of GDF8 in cattle (Ashmore et al., 1974,Growth, 38:501-507; Swatland and Kieffer, J. Anim. Sci., 1994,38:752-757; McPhenton and Lee, Proc. Natl. Acad. Sci. USA, 1997,94:12457-12461; and Kambadur et al., Genome Res., 1997, 7:910-915) and,strikingly, in humans (Schuelke et al., N Engl J Med 2004; 350:2682-8).Studies have also shown that muscle wasting associated withHIV-infection in humans is accompanied by increases in GDF8 proteinexpression (Gonzalez-Cadavid et al., PNAS, 1998, 95:14938-43). Inaddition, GDF8 can modulate the production of muscle-specific enzymes(e.g., creatine kinase) and modulate myoblast cell proliferation (WO00/43781). The GDF8 propeptide can noncovalently bind to the mature GDF8domain dimer, inactivating its biological activity (Miyazono et al.(1988) J. Biol. Chem., 263: 6407-6415; Wakefield et al. (1988) J. Biol.Chem., 263; 7646-7654; and Brown et al. (1990) Growth Factors, 3:35-43). Other proteins which bind to GDF8 or structurally relatedproteins and inhibit their biological activity include follistatin, andpotentially, follistatin-related proteins (Gamer et al. (1999) Dev.Biol., 208: 222-232).

Growth and Differentiation Factor-11 (GDF11), also known as BMP11, is asecreted protein (McPherron et al., 1999, Nat. Genet. 22: 260-264).GDF11 is expressed in the tail bud, limb bud, maxillary and mandibulararches, and dorsal root ganglia during mouse development (Nakashima etal., 1999, Mech. Dev. 80: 185-189). GDF11 plays a unique role inpatterning both mesodermal and neural tissues (Gamer et al., 1999, DevBiol., 208:222-32). GDF11 was shown to be a negative regulator ofchondrogenesis and myogenesis in developing chick limb (Gamer et al.,2001, Dev Biol. 229:407-20). The expression of GDF11 in muscle alsosuggests its role in regulating muscle growth in a similar way to GDF8.In addition, the expression of GDF11 in brain suggests that GDF11 mayalso possess activities that relate to the function of the nervoussystem. Interestingly, GDF11 was found to inhibit neurogenesis in theolfactory epithelium (Wu et al., 2003, Neuron. 37:197-207). Hence, GDF11may have in vitro and in vivo applications in the treatment of diseasessuch as muscle diseases and neurodegenerative diseases (e.g.,amyotrophic lateral sclerosis).

In certain aspects, the present invention relates to the use of certainActRIIB polypeptides to antagonize the signaling of ActRIIB ligandsgenerally, in any process associated with ActRIIB activity. Optionally.ActRIIB polypeptides of the invention may antagonize one or more ligandsof ActRIIB receptors, such as activin, Nodal, GDF8, GDF11, and BMP7, andmay therefore be useful in the treatment of additional disorders.

Therefore, the present invention contemplates using ActRIIB polypeptidesin treating or preventing diseases or conditions that are associatedwith abnormal activity of an ActRIIB or an ActRIIB ligand. ActRIIB orActRIIB ligands are involved in the regulation of many criticalbiological processes. Due to their key functions in these processes,they may be desirable targets for therapeutic intervention. For example,ActRIIB polypeptides (e.g., ActRIIB-Fc polypeptides) may be used totreat human or animal disorders or conditions. Example of such disordersor conditions include, but are not limited to, metabolic disorders suchas type 2 diabetes, impaired glucose tolerance, metabolic syndrome(e.g., syndrome X), and insulin resistance induced by trauma (e.g.,burns or nitrogen imbalance); adipose tissue disorders (e.g., obesity);muscle and neuromuscular disorders such as muscular dystrophy (includingDuchenne muscular dystrophy); amyotrophic lateral sclerosis (ALS);muscle atrophy; organ atrophy; frailty; carpal tunnel syndrome;congestive obstructive pulmonary disease; and sarcopenia, cachexia andother muscle wasting syndromes. Other examples include osteoporosis,especially in the elderly and/or postmenopausal women;glucocorticoid-induced osteoporosis; osteopenia; osteoarthritis; andosteoporosis-related fractures. Yet further examples include low bonemass due to chronic glucocorticoid therapy, premature gonadal failure,androgen suppression, vitamin D deficiency, secondaryhyperparathyroidism, nutritional deficiencies, and anorexia nervosa.These disorders and conditions are discussed below under “ExemplaryTherapeutic Uses.” As noted, the truncated ActRIIB polypeptidesdisclosed herein appear to have particularly beneficial effects onmetabolic parameters.

The terms used in this specification generally have their ordinarymeanings in the art, within the context of this invention and in thespecific context where each term is used. Certain terms are discussedbelow or elsewhere in the specification, to provide additional guidanceto the practitioner in describing the compositions and methods of theinvention and how to make and use them. The scope or meaning of any useof a term will be apparent from the specific context in which the termis used.

“About” and “approximately” shall generally mean an acceptable degree oferror for the quantity measured given the nature or precision of themeasurements. Typically, exemplary degrees of error are within 20percent (%), preferably within 10%, and more preferably within 5% of agiven value or range of values.

Alternatively, and particularly in biological systems, the terms “about”and “approximately” may mean values that are within an order ofmagnitude, preferably within 5-fold and more preferably within 2-fold ofa given value. Numerical quantities given herein are approximate unlessstated otherwise, meaning that the term “about” or “approximately” canbe inferred when not expressly stated.

The methods of the invention may include steps of comparing sequences toeach other, including wild-type sequence to one or more mutants(sequence variants). Such comparisons typically comprise alignments ofpolymer sequences, e.g., using sequence alignment programs and/oralgorithms that are well known in the art (for example, BLAST, FASTA andMEGALIGN, to name a few). The skilled artisan can readily appreciatethat, in such alignments, where a mutation contains a residue insertionor deletion, the sequence alignment will introduce a “gap” (typicallyrepresented by a dash, or “A”) in the polymer sequence not containingthe inserted or deleted residue.

“Homologous,” in all its grammatical forms and spelling variations,refers to the relationship between two proteins that possess a “commonevolutionary origin,” including proteins from superfamilies in the samespecies of organism, as well as homologous proteins from differentspecies of organism. Such proteins (and their encoding nucleic acids)have sequence homology, as reflected by their sequence similarity,whether in terms of percent identity or by the presence of specificresidues or motifs and conserved positions.

The term “sequence similarity,” in all its grammatical forms, refers tothe degree of identity or correspondence between nucleic acid or aminoacid sequences that may or may not share a common evolutionary origin.

However, in common usage and in the instant application, the term“homologous,” when modified with an adverb such as “highly,” may referto sequence similarity and may or may not relate to a commonevolutionary origin.

2. ActRIIB Polypeptides

In certain aspects, the invention relates to ActRIIB polypeptides (e.g.,ActRIIB-Fc polypeptides), and particularly truncated forms exemplifiedby polypeptides comprising amino acids 25-131 of SEQ ID NO:1, andvariants thereof. Optionally, the fragments, functional variants, andmodified forms have similar or the same biological activities of theircorresponding wild-type ActRIIB polypeptides. For example, an ActRIIBvariant of the invention may bind to and inhibit function of an ActRIIBligand (e.g., activin A, activin AB, activin B, Nodal, GDF8, GDF11 orBMP7). Optionally, an ActRIIB polypeptide modulates growth of tissuessuch as bone, cartilage, muscle or fat or metabolic parameters such astriglycerides, free fatty acids or insulin. Examples of ActRIIBpolypeptides include human ActRIIB precursor polypeptide (SEQ ID NO: 1),and Fc fusion proteins, e.g., SEQ ID NOS. 3 and 8. Variations on thesepolypeptides may be prepared according to the following guidance. Thenumbering of amino acids in the ActRIIB polypeptides is based on thesequence of SEQ ID NO: 1, regardless of whether the native leadersequence is used.

The disclosure identifies functionally active portions and variants ofActRIIB. Applicants have ascertained that an Fc fusion protein havingthe sequence disclosed by Hilden et al. (Blood. 1994 Apr. 15;83(8):2163-70), which has an Alanine at the position corresponding toamino acid 64 of SEQ ID NO: 1 (A64), has a relatively low affinity foractivin and GDF-11. By contrast, the same Fc fusion protein with anArginine at position 64 (R64) has an affinity for activin and GDF-11 inthe low nanomolar to high picomolar range. Therefore, a sequence with anR64 is used as the wild-type reference sequence for human ActRIIB inthis disclosure.

Attisano et al. (Cell. 1992 Jan. 10; 68(1):97-108) showed that adeletion of the proline knot at the C-terminus of the extracellulardomain of ActRIIB reduced the affinity of the receptor for activin.Mutations of P129 and P130 do not substantially decrease ligand binding.

The ActRIIB ligand binding pocket is defined by residues Y31, N33, N35,L38 through T41, E47, E50, Q53 through K55, L57, H58, Y60, S62, K74, W78through N83, Y85, R87, A92, and E94 through F101. At these positions, itis expected that conservative mutations will be tolerated, although aK74A mutation is well-tolerated, as are R40A, K55A, F82A and mutationsat position L79. R40 is a K in Xenopus, indicating that basic aminoacids at this position will be tolerated. Q53 is R in bovine ActRIIB andK in Xenopus ActRIIB, and therefore amino acids including R, K, Q, N andH will be tolerated at this position. Thus, an ActRIIB protein may beone that comprises amino acids 25-131 and comprising no more than 1, 2,5, 10 or 15 conservative amino acid changes in the ligand bindingpocket, and zero, one or more non-conservative alterations at positions40, 53, 55, 74, 79 and/or 82 in the ligand binding pocket. Such aprotein may retain greater than 80%, 90%, 95% or 99% sequence identityto the sequence of amino acids 25-131 of SEQ ID NO:1. Sites outside thebinding pocket, at which variability may be particularly well tolerated,include the amino and carboxy termini of the extracellular domain (asnoted above), and positions 42-46 and 65-73. An asparagine to alaninealteration at position 65 (N65A) actually improves ligand binding in theA64 background, and is thus expected to have no detrimental effect onligand binding in the R64 background. This change probably eliminatesglycosylation at N65 in the A64 background, thus demonstrating that asignificant change in this region is likely to be tolerated. While anR64A change is poorly tolerated, R64K is well-tolerated, and thusanother basic residue, such as H may be tolerated at position 64.

ActRIIB is well-conserved across nearly all vertebrates, with largestretches of the extracellular domain conserved completely. Many of theligands that bind to ActRIIB are also highly conserved. Accordingly,comparisons of ActRIIB sequences from various vertebrate organismsprovide insights into residues that may be altered. Therefore, anactive, human ActRIIB may include one or more amino acids atcorresponding positions from the sequence of another vertebrate ActRIIB,or may include a residue that is similar to that in the human or othervertebrate sequence. The following examples illustrate this approach todefining an active ActRIIB variant. L46 is a valine in Xenopus ActRIIB,and so this position may be altered, and optionally may be altered toanother hydrophobic residue, such as V, I or F, or a non-polar residuesuch as A. E52 is a K in Xenopus, indicating that this site may betolerant of a wide variety of changes, including polar residues, such asE, D, K, R, H, S, T, P, G, Y and probably A. T93 is a K in Xenopus,indicating that a wide structural variation is tolerated at thisposition, with polar residues favored, such as S, K, R, E, D, H, G, P, Gand Y. F108 is a Y in Xenopus, and therefore Y or other hydrophobicgroup, such as I, V or L should be tolerated. E111 is K in Xenopus,indicating that charged residues will be tolerated at this position,including D, R, K and H, as well as Q and N. R112 is K in Xenopus,indicating that basic residues are tolerated at this position, includingR and H. A at position 119 is relatively poorly conserved, and appearsas P in rodents and V in Xenopus, thus essentially any amino acid shouldbe tolerated at this position.

Further N-linked glycosylation sites (N-X-S/T) may be added to anActRIIB polypeptide, and may increase the serum half-life of anActRIIB-Fc fusion protein, relative to the ActRIIB(R64)-Fc form.Examples of NX(T/S) sequences are found at 42-44 (NQS) and 65-67 (NSS),although the latter may not be efficiently glycosylated with the R atposition 64. N-X-S/T sequences may be generally introduced at positionsoutside the ligand binding pocket. Particularly suitable sites for theintroduction of non-endogenous N-X-S/T sequences include amino acids20-29, 20-24, 22-25, 109-134, 120-134 or 129-134. N-X-SIT sequences mayalso be introduced into the linker between the ActRIIB sequence and theFc or other fusion component. Such a site may be introduced with minimaleffort by introducing an N in the correct position with respect to apre-existing S or T, or by introducing an S or T at a positioncorresponding to a pre-existing N. Thus, desirable alterations thatwould create an N-linked glycosylation site are: A24N, R64N, S67N(possibly combined with an N65A alteration), E106N, R112N, G120N, E123N,P129N, A132N, R112S and R112T. Any S that is predicted to beglycosylated may be altered to a T without creating an immunogenic site,because of the protection afforded by the glycosylation. Likewise, any Tthat is predicted to be glycosylated may be altered to an S. Thus thealterations S67T and S44T are contemplated. Likewise, in an A24Nvariant, an S26T alteration may be used. Accordingly, an ActRIIB variantmay include one or more additional, non-endogenous N-linkedglycosylation consensus sequences.

The variations described may be combined in various ways. Additionally,there are amino acid positions in ActRIIB that are often beneficial toconserve. These include position 64 (basic amino acid), position 80(acidic or hydrophobic amino acid), position 78 (hydrophobic, andparticularly tryptophan), position 37 (acidic, and particularly asparticor glutamic acid), position 56 (basic amino acid), position 60(hydrophobic amino acid, particularly phenylalanine or tyrosine). Otherpositions that may be desirable to conserve are as follows: position 52(acidic amino acid), position 55 (basic amino acid), position 81(acidic), 98 (polar or charged, particularly E, D, R or K).

In certain embodiments, the present invention contemplates makingfunctional variants by modifying the structure of an ActRIIB polypeptidefor such purposes as enhancing therapeutic efficacy, or stability (e.g.,ex vivo shelf life and resistance to proteolytic degradation in vivo).Modified ActRIIB polypeptides can also be produced, for instance, byamino acid substitution, deletion, or addition. For instance, it isreasonable to expect that an isolated replacement of a leucine with anisoleucine or valine, an aspartate with a glutamate, a threonine with aserine, or a similar replacement of an amino acid with a structurallyrelated amino acid (e.g., conservative mutations) will not have a majoreffect on the biological activity of the resulting molecule.Conservative replacements are those that take place within a family ofamino acids that are related in their side chains. Whether a change inthe amino acid sequence of an ActRIIB polypeptide results in afunctional homolog can be readily determined by assessing the ability ofthe variant ActRIIB polypeptide to produce a response in cells in afashion similar to the wild-type ActRIIB polypeptide, or to bind to oneor more ligands, such as activin, GDF-11 or myostatin in a fashionsimilar to wild type.

In certain specific embodiments, the present invention contemplatesmaking mutations in the extracellular domain (also referred to asligand-binding domain) of an ActRIIB polypeptide such that the variant(or mutant) ActRIIB polypeptide has altered ligand-binding activities(e.g., binding affinity or binding specificity). In certain cases, suchvariant ActRIIB polypeptides have altered (elevated or reduced) bindingaffinity for a specific ligand. In other cases, the variant ActRIIBpolypeptides have altered binding specificity for their ligands.

In certain embodiments, the present invention contemplates specificmutations of the ActRIIB polypeptides so as to alter the glycosylationof the polypeptide. Such mutations may be selected so as to introduce oreliminate one or more glycosylation sites, such as O-linked or N-linkedglycosylation sites. Asparagine-linked glycosylation recognition sitesgenerally comprise a tripeptide sequence, asparagine-X-threonine (where“X” is any amino acid) which is specifically recognized by appropriatecellular glycosylation enzymes. The alteration may also be made by theaddition of, or substitution by, one or more serine or threonineresidues to the sequence of the wild-type ActRIIB polypeptide (forO-linked glycosylation sites). A variety of amino acid substitutions ordeletions at one or both of the first or third amino acid positions of aglycosylation recognition site (and/or amino acid deletion at the secondposition) results in non-glycosylation at the modified tripeptidesequence. Another means of increasing the number of carbohydratemoieties on an ActRIIB polypeptide is by chemical or enzymatic couplingof glycosides to the ActRIIB polypeptide. Depending on the coupling modeused, the sugar(s) may be attached to (a) arginine and histidine; (b)free carboxyl groups; (c) free sulfhydryl groups such as those ofcysteine; (d) free hydroxyl groups such as those of serine, threonine,or hydroxyproline; (e) aromatic residues such as those of phenylalanine,tyrosine, or tryptophan; or (f) the amide group of glutamine. Thesemethods are described in WO 87/05330 published Sep. 11, 1987, and inAplin and Wriston (1981) CRC Crit. Rev. Biochem., pp. 259-306,incorporated by reference herein. Removal of one or more carbohydratemoieties present on an ActRIIB polypeptide may be accomplishedchemically and/or enzymatically. Chemical deglycosylation may involve,for example, exposure of the ActRIIB polypeptide to the compoundtrifluoromethanesulfonic acid, or an equivalent compound. This treatmentresults in the cleavage of most or all sugars except the linking sugar(N-acetylglucosamine or N-acetylgalactosamine), while leaving the aminoacid sequence intact. Chemical deglycosylation is further described byHakimuddin et al. (1987) Arch. Biochem. Biophys. 259:52 and by Edge etal. (1981) Anal. Biochem. 118:131. Enzymatic cleavage of carbohydratemoieties on ActRIIB polypeptides can be achieved by the use of a varietyof endo- and exo-glycosidases as described by Thotakura et al. (1987)Meth. Enzymol. 138:350. The sequence of an ActRIIB polypeptide may beadjusted, as appropriate, depending on the type of expression systemused, as mammalian, yeast, insect and plant cells may all introducediffering glycosylation patterns that can be affected by the amino acidsequence of the peptide. In general, ActRIIB proteins for use in humanswill be expressed in a mammalian cell line that provides properglycosylation, such as HEK293 or CHO cell lines, although othermammalian expression cell lines are expected to be useful as well.

This disclosure further contemplates a method of generating variants,particularly sets of combinatorial variants of an ActRIIB polypeptide,including, optionally, truncation variants; pools of combinatorialmutants are especially useful for identifying functional variantsequences. The purpose of screening such combinatorial libraries may beto generate, for example, ActRIIB polypeptide variants which havealtered properties, such as altered pharmacokinetics, or altered ligandbinding. A variety of screening assays are provided below, and suchassays may be used to evaluate variants. For example, an ActRIIBpolypeptide variant may be screened for ability to bind to an ActRIIBpolypeptide, to prevent binding of an ActRIIB ligand to an ActRIIBpolypeptide.

The activity of an ActRIIB polypeptide or its variants may also betested in a cell-based or in vivo assay. For example, the effect of anActRIIB polypeptide variant on the expression of genes involved in boneproduction in an osteoblast or precursor may be assessed. This may, asneeded, be performed in the presence of one or more recombinant ActRIIBligand protein (e.g., BMP7), and cells may be transfected so as toproduce an ActRIIB polypeptide and/or variants thereof, and optionally,an ActRIIB ligand. Likewise, an ActRIIB polypeptide may be administeredto a mouse or other animal, and one or more bone properties, such asdensity or volume may be assessed. The healing rate for bone fracturesmay also be evaluated. Similarly, the activity of an ActRIIB polypeptideor its variants may be tested in muscle cells, adipocytes, and neuronalcells for any effect on growth of these cells, for example, by theassays as described below. Such assays are well known and routine in theart. A SMAD-responsive reporter gene may be used in such cell lines tomonitor effects on downstream signaling.

Combinatorially-derived variants can be generated which have a selectivepotency relative to a naturally occurring ActRIIB polypeptide. Suchvariant proteins, when expressed from recombinant DNA constructs, can beused in gene therapy protocols. Likewise, mutagenesis can give rise tovariants which have intracellular half-lives dramatically different thanthe corresponding a wild-type ActRIIB polypeptide. For example, thealtered protein can be rendered either more stable or less stable toproteolytic degradation or other processes which result in destructionof, or otherwise inactivation of a native ActRIIB polypeptide. Suchvariants, and the genes which encode them, can be utilized to alterActRIIB polypeptide levels by modulating the half-life of the ActRIIBpolypeptides. For instance, a short half-life can give rise to moretransient biological effects and, when part of an inducible expressionsystem, can allow tighter control of recombinant ActRIIB polypeptidelevels within the cell.

In certain embodiments, the ActRIIB polypeptides of the invention mayfurther comprise post-translational modifications in addition to anythat are naturally present in the ActRIIB polypeptides. Suchmodifications include, but are not limited to, acetylation,carboxylation, glycosylation, phosphorylation, lipidation, andacylation. As a result, the modified ActRIIB polypeptides may containnon-amino acid elements, such as polyethylene glycols, lipids, poly- ormono-saccharide, and phosphates. Effects of such non-amino acid elementson the functionality of a ActRIIB polypeptide may be tested as describedherein for other ActRIIB polypeptide variants. When an ActRIIBpolypeptide is produced in cells by cleaving a nascent form of theActRIIB polypeptide, post-translational processing may also be importantfor correct folding and/or function of the protein. Different cells(such as CHO, HeLa, MDCK, 293, WI38, NIH-3T3 or HEK293) have specificcellular machinery and characteristic mechanisms for suchpost-translational activities and may be chosen to ensure the correctmodification and processing of the ActRIIB polypeptides.

In certain aspects, functional variants or modified forms of the ActRIIBpolypeptides include fusion proteins having at least a portion of theActRIIB polypeptides and one or more fusion domains. Well known examplesof such fusion domains include, but are not limited to, polyhistidine,Glu-Glu, glutathione S transferase (GST), thioredoxin, protein A,protein G, an immunoglobulin heavy chain constant region (e.g., an Fc),maltose binding protein (MBP), or human serum albumin. A fusion domainmay be selected so as to confer a desired property. For example, somefusion domains are particularly useful for isolation of the fusionproteins by affinity chromatography. For the purpose of affinitypurification, relevant matrices for affinity chromatography, such asglutathione-, amylase-, and nickel- or cobalt-conjugated resins areused. Many of such matrices are available in “kit” form, such as thePharmacia GST purification system and the QIAexpress™ system (Qiagen)useful with (HIS₆) fusion partners. As another example, a fusion domainmay be selected so as to facilitate detection of the ActRIIBpolypeptides. Examples of such detection domains include the variousfluorescent proteins (e.g., GFP) as well as “epitope tags,” which areusually short peptide sequences for which a specific antibody isavailable. Well known epitope tags for which specific monoclonalantibodies are readily available include FLAG, influenza virushaemagglutinin (HA), and c-myc tags. In some cases, the fusion domainshave a protease cleavage site, such as for Factor Xa or Thrombin, whichallows the relevant protease to partially digest the fusion proteins andthereby liberate the recombinant proteins therefrom. The liberatedproteins can then be isolated from the fusion domain by subsequentchromatographic separation. In certain preferred embodiments, an ActRIIBpolypeptide is fused with a domain that stabilizes the ActRIIBpolypeptide in vivo (a “stabilizer” domain). By “stabilizing” is meantanything that increases serum half life, regardless of whether this isbecause of decreased destruction, decreased clearance by the kidney, orother pharmacokinetic effect Fusions with the Fc portion of aninmmunoglobulin are known to confer desirable pharmacokinetic propertieson a wide range of proteins. Likewise, fusions to human serum albumincan confer desirable properties. Other types of fusion domains that maybe selected include multimerizing (e.g., dimerizing, tetramerizing)domains and functional domains (that confer an additional biologicalfunction, such as further stimulation of muscle growth).

As a specific example, the present invention provides a fusion proteinas a GDF8 antagonist which comprises an extracellular (e.g.,GDF8-binding) domain fused to an Fc domain (e.g., SEQ ID NO: 9).

THTCPPCPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVD(A)VSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCK(A)VSNKALPVPIEKTISKAKGRPREPQVYTLPPSREEMTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGPFFLYSKLTVDKSRWQQGNVFSCSVMHEALHN(A)HYTQKSLSLSPGK*

Optionally, the Fc domain has one or more mutations at residues such asAsp-265, lysine 322, and Asn-434. In certain cases, the mutant Fc domainhaving one or more of these mutations (e.g., Asp-265 mutation) hasreduced ability of binding to the Fcγ receptor relative to a wildtype Fcdomain. In other cases, the mutant Fc domain having one or more of thesemutations (e.g., Asn-434 mutation) has increased ability of binding tothe MHC class I-related Fc-receptor (FcRN) relative to a wildtype Fcdomain.

It is understood that different elements of the fusion proteins may bearranged in any manner that is consistent with the desiredfunctionality. For example, an ActRIIB polypeptide may be placedC-terminal to a heterologous domain, or, alternatively, a heterologousdomain may be placed C-terminal to an ActRIIB polypeptide. The ActRIIBpolypeptide domain and the heterologous domain need not be adjacent in afusion protein, and additional domains or amino acid sequences may beincluded C- or N-terminal to either domain or between the domains.

In certain embodiments, the ActRIIB polypeptides of the presentinvention contain one or more modifications that are capable ofstabilizing the ActRIIB polypeptides. For example, such modificationsenhance the in vitro half life of the ActRIIB polypeptides, enhancecirculatory half life of the ActRIIB polypeptides or reducingproteolytic degradation of the ActRIIB polypeptides. Such stabilizingmodifications include, but are not limited to, fusion proteins(including, for example, fusion proteins comprising an ActRIIBpolypeptide and a stabilizer domain), modifications of a glycosylationsite (including, for example, addition of a glycosylation site to anActRIIB polypeptide), and modifications of carbohydrate moiety(including, for example, removal of carbohydrate moieties from anActRIIB polypeptide). In the case of fusion proteins, an ActRIIBpolypeptide is fused to a stabilizer domain such as an IgG molecule(e.g., an Fc domain). As used herein, the term “stabilizer domain” notonly refers to a fusion domain (e.g., Fc) as in the case of fusionproteins, but also includes nonproteinaceous modifications such as acarbohydrate moiety, or nonproteinaceous polymer, such as polyethyleneglycol.

In certain embodiments, the present invention makes available isolatedand/or purified forms of the ActRIIB polypeptides, which are isolatedfrom, or otherwise substantially free of, other proteins.

In certain embodiments, ActRIIB polypeptides (unmodified or modified) ofthe invention can be produced by a variety of art-known techniques. Forexample, such ActRIIB polypeptides can be synthesized using standardprotein chemistry techniques such as those described in Bodansky. M.Principles of Peptide Synthesis, Springer Verlag, Berlin (1993) andGrant G. A. (ed.), Synthetic Peptides: A User's Guide, W. H. Freeman andCompany, New York (1992). In addition, automated peptide synthesizersare commercially available (e.g., Advanced ChemTech Model 396;Milligen/Biosearch 9600). Alternatively, the ActRIIB polypeptides,fragments or variants thereof may be recombinantly produced usingvarious expression systems (e.g., E. coli, Chinese Hamster Ovary cells,COS cells, baculovirus) as is well known in the art (also see below). Ina further embodiment, the modified or unmodified ActRIIB polypeptidesmay be produced by digestion of naturally occurring or recombinantlyproduced full-length ActRIIB polypeptides by using, for example, aprotease, e.g., trypsin, thermolysin, chymotrypsin, pepsin, or pairedbasic amino acid converting enzyme (PACE). Computer analysis (using acommercially available software, e.g., MacVector, Omega, PCGene,Molecular Simulation, Inc.) can be used to identify proteolytic cleavagesites. Alternatively, such ActRIIB polypeptides may be produced fromnaturally occurring or recombinantly produced full-length ActRIIBpolypeptides such as standard techniques known in the art, such as bychemical cleavage (e.g., cyanogen bromide, hydroxylamine).

3. Nucleic Acids Encoding ActRIIB Polypeptides

In certain aspects, the invention provides isolated and/or recombinantnucleic acids encoding any of the ActRIIB polypeptides disclosed herein.For example, SEQ ID NO: 4 encodes an ActRIIB(25-131)-hFc precursorpolypeptide, while SEQ ID NO: 6 encodes a the same protein but with analternative sequence, and nucleotides 73-396 of each of SEQ ID NOS: 4and 6 encode the ActRIIB-derived portion of the encoded proteins. Thesubject nucleic acids may be single-stranded or double stranded Suchnucleic acids may be DNA or RNA molecules. These nucleic acids are maybe used, for example, in methods for making ActRIIB polypeptides.

For example, the following sequence encodes a naturally occurring humanActRIIB precursor polypeptide (SEQ ID NO: 2) (nucleotides 5-1543 ofNM_001106, 1539 bp):

atgacggcgccctgggtggccctcgccctcctctggggatcgctgtggcccggctctgggcgtggggaggctgagacacgggagtgcatctactacaacgccaactgggagctggagcgcaccaaccagagcggcctggagcgctgcgaaggcgagcaggacaagcggctgcactgctacgcctcctggcgcaacagctctggcaccatcgagctcgtgaagaagggctgctggctagatgacttcaactgctacgataggcaggagtgtgtggccactgaggagaacccccaggtgtacttctgctgctgtgaaggcaacttctgcaacgagcgcttcactcatttgccagaggctgggggcccggaagtcacgtacgagccacccccgacagcccccaccctgctcacggtgctggcctactcactgctgcccatcgggggcctttccctcatcgtcctgctggccttttggatgtaccggcatcgcaagcccccctacggtcatgtggacatccatgaggaccctgggcctccaccaccatcccctctggtgggcctgaagccactgcagctgctggagatcaaggctcgggggcgctttggctgtgtctggaaggcccagctcatgaatgactttgtagctgtcaagatcttcccactccaggacaagcagtcgtggcagagtgaacgggagatcttcagcacacctggcatgaagcacgagaacctgctacagttcattgctgccgagaagcgaggctccaacctcgaagtagagctgtggctcatcacggccttccatgacaagggctccctcacggattacctcaaggggaacatcatcacatggaacgaactgtgtcatgtagcagagacgatgtcacgaggcctctcatacctgcatgaggatgtgccctggtgccgtggcgagggccacaagccgtctattgcccacagggactttaaaagtaagaatgtattgctgaagagcgacctcacagccgtgctggctgactttggcttggctgttcgatttgagccagggaaacctccaggggacacccacggacaggtaggcacgagacggtacatggctcctgaggtgctcgagggagccatcaacttccagagagatgccttcctgcgcattgacatgtatgccatggggttggtgctgtgggagcttgtgtctcgctgcaaggctgcagacggacccgtggatgagtacatgctgccctttgaggaagagattggccagcacccttcgttggaggagctgcaggaggtggtggtgcacaagaagatgaggcccaccattaaagatcactggttgaaacacccgggcctggcccagctttgtgtgaccatcgaggagtgctgggaccatgatgcagaggctcgcttgtccgcgggctgtgtggaggagcgggtgtccctgattcggaggtcggtcaacggcactacctcggactgtctcgtttccctggtgacctctgtcaccaatgtggacctgcccc ctaaagagtcaagcatctaa

The following sequence encodes a human soluble (extracellular) ActRIIBpolypeptide (SEQ ID NO: 10) (348 bp).

tctgggcgtggggaggctgagacacgggagtgcatctactacaacgccaactgggagctggagcgcaccaaccagagcggcctggagcgctgcgaaggcgagcaggacaagcggctgcactgctacgcctcctggcgcaacagctctggcaccatcgagctcgtgaagaagggctgctggctagatgacttcaactgctacgataggcaggagtgtgtggccactgaggagaacccccaggtgtacttctgctgctgtgaaggcaacttctgcaacgagcgcttcactcatttgccagaggctgggggcccggaagtcacgtacgagccacccccgacagcccccac

In certain aspects, the subject nucleic acids encoding ActRIIBpolypeptides are further understood to include nucleic acids that arevariants of SEQ ID NO: 4 or 6. Variant nucleotide sequences includesequences that differ by one or more nucleotide substitutions, additionsor deletions, such as allelic variants; and will, therefore, includecoding sequences that differ from the nucleotide sequence of the codingsequence designated in SEQ ID NO: 4 or 6.

In certain embodiments, the invention provides isolated or recombinantnucleic acid sequences that are at least 80%, 85%, 90%, 95%, 97%, 98%,99% or 100% identical to SEQ ID NO: 4 or 6, and particularly thoseportions thereof that are derived from ActRIIB (nucleotides 73-396). Oneof ordinary skill in the art will appreciate that nucleic acid sequencescomplementary to SEQ ID NO: 4 or 6, and variants of SEQ ID NO: 4 arealso within the scope of this invention. In further embodiments, thenucleic acid sequences of the invention can be isolated, recombinant,and/or fused with a heterologous nucleotide sequence, or in a DNAlibrary.

In other embodiments, nucleic acids of the invention also includenucleotide sequences that hybridize under highly stringent conditions tothe nucleotide sequence designated in SEQ ID NO: 4 or 6, complementsequence of SEQ ID NO: 4 or 6, or fragments thereof (e.g., nucleotides73-396). As discussed above, one of ordinary skill in the art willunderstand readily that appropriate stringency conditions which promoteDNA hybridization can be varied. One of ordinary skill in the art willunderstand readily that appropriate stringency conditions which promoteDNA hybridization can be varied. For example, one could perform thehybridization at 6.0× sodium chloride/sodium citrate (SSC) at about 45°C., followed by a wash of 2.0×SSC at 50° C. For example, the saltconcentration in the wash step can be selected from a low stringency ofabout 2.0×SSC at 50° C. to a high stringency of about 0.2×SSC at 50° C.In addition, the temperature in the wash step can be increased from lowstringency conditions at room temperature, about 22° C., to highstringency conditions at about 65° C. Both temperature and salt may bevaried, or temperature or salt concentration may be held constant whilethe other variable is changed. In one embodiment, the invention providesnucleic acids which hybridize under low stringency conditions of 6×SSCat room temperature followed by a wash at 2×SSC at room temperature.

Isolated nucleic acids which differ from the nucleic acids as set forthin SEQ ID NO: 4 or 6 due to degeneracy in the genetic code are alsowithin the scope of the invention. For example, a number of amino acidsare designated by more than one triplet. Codons that specify the sameamino acid, or synonyms (for example, CAU and CAC are synonyms forhistidine) may result in “silent” mutations which do not affect theamino acid sequence of the protein. However, it is expected that DNAsequence polymorphisms that do lead to changes in the amino acidsequences of the subject proteins will exist among mammalian cells. Oneskilled in the art will appreciate that these variations in one or morenucleotides (up to about 3-5% of the nucleotides) of the nucleic acidsencoding a particular protein may exist among individuals of a givenspecies due to natural allelic variation. Any and all such nucleotidevariations and resulting amino acid polymorphisms are within the scopeof this invention.

In certain embodiments, the recombinant nucleic acids of the inventionmay be operably linked to one or more regulatory nucleotide sequences inan expression construct. Regulatory nucleotide sequences will generallybe appropriate to the host cell used for expression. Numerous types ofappropriate expression vectors and suitable regulatory sequences areknown in the art for a variety of host cells. Typically, said one ormore regulatory nucleotide sequences may include, but are not limitedto, promoter sequences, leader or signal sequences, ribosomal bindingsites, transcriptional start and termination sequences, translationalstart and termination sequences, and enhancer or activator sequences.Constitutive or inducible promoters as known in the art are contemplatedby the invention. The promoters may be either naturally occurringpromoters, or hybrid promoters that combine elements of more than onepromoter. An expression construct may be present in a cell on anepisome, such as a plasmid, or the expression construct may be insertedin a chromosome. In a preferred embodiment, the expression vectorcontains a selectable marker gene to allow the selection of transformedhost cells. Selectable marker genes are well known in the art and willvary with the host cell used.

In certain aspects of the invention, the subject nucleic acid isprovided in an expression vector comprising a nucleotide sequenceencoding an ActRIIB polypeptide and operably linked to at least oneregulatory sequence. Regulatory sequences are art-recognized and areselected to direct expression of the ActRIIB polypeptide. Accordingly,the term regulatory sequence includes promoters, enhancers, and otherexpression control elements. Exemplary regulatory sequences aredescribed in Goeddel; Gene Expression Technology: Methods in Enzymology,Academic Press. San Diego, Calif. (1990). For instance, any of a widevariety of expression control sequences that control the expression of aDNA sequence when operatively linked to it may be used in these vectorsto express DNA sequences encoding an ActRIIB polypeptide. Such usefulexpression control sequences, include, for example, the early and latepromoters of SV40, tet promoter, adenovirus or cytomegalovirus immediateearly promoter, RSV promoters, the lac system, the tip system, the TACor TRC system, T7 promoter whose expression is directed by T7 RNApolymerase, the major operator and promoter regions of phage lambda, thecontrol regions for fd coat protein, the promoter for 3-phosphoglyceratekinase or other glycolytic enzymes, the promoters of acid phosphatase,e.g., Pho5, the promoters of the yeast α-mating factors, the polyhedronpromoter of the baculovirus system and other sequences known to controlthe expression of genes of prokaryotic or eukaryotic cells or theirviruses, and various combinations thereof. It should be understood thatthe design of the expression vector may depend on such factors as thechoice of the host cell to be transformed and/or the type of proteindesired to be expressed. Moreover, the vector's copy number, the abilityto control that copy number and the expression of any other proteinencoded by the vector, such as antibiotic markers, should also beconsidered.

A recombinant nucleic acid of the invention can be produced by ligatingthe cloned gene, or a portion thereof, into a vector suitable forexpression in either prokaryotic cells, eukaryotic cells (yeast, avian,insect or mammalian), or both. Expression vehicles for production of arecombinant ActRIIB polypeptide include plasmids and other vectors. Forinstance, suitable vectors include plasmids of the types: pBR322-derivedplasmids, pEMBL-derived plasmids, pEX-derived plasmids, pBTac-derivedplasmids and pUC-derived plasmids for expression in prokaryotic cells,such as E. coli.

Some mammalian expression vectors contain both prokaryotic sequences tofacilitate the propagation of the vector in bacteria, and one or moreeukaryotic transcription units that are expressed in eukaryotic cells.The pcDNAI/amp, pcDNAI/neo, pRc/CMV, pSV2gpt, pSV2neo, pSV2-dhfr, pTk2,pRSVneo, pMSG, pSVT7, pko-neo and pHyg derived vectors are examples ofmammalian expression vectors suitable for transfection of eukaryoticcells. Some of these vectors are modified with sequences from bacterialplasmids, such as pBR322, to facilitate replication and drug resistanceselection in both prokaryotic and eukaryotic cells. Alternatively,derivatives of viruses such as the bovine papilloma virus (BPV-1), orEpstein-Barr virus (pHEBo, pREP-derived and p205) can be used fortransient expression of proteins in eukaryotic cells. Examples of otherviral (including retroviral) expression systems can be found below inthe description of gene therapy delivery systems. The various methodsemployed in the preparation of the plasmids and in transformation ofhost organisms are well known in the art. For other suitable expressionsystems for both prokaryotic and eukaryotic cells, as well as generalrecombinant procedures, see Molecular Cloning A Laboratory Manual, 2ndEd., ed. by Sambrook, Fritsch and Maniatis (Cold Spring HarborLaboratory Press, 1989) Chapters 16 and 17. In some instances, it may bedesirable to express the recombinant polypeptides by the use of abaculovirus expression system. Examples of such baculovirus expressionsystems include pVL-derived vectors (such as pVL1392, pVL1393 andpVL941), pAcUW-derived vectors (such as pAcUW1), and pBlueBac-derivedvectors (such as the β-gal containing pBheBac III).

In a preferred embodiment, a vector will be designed for production ofthe subject ActRIIB polypeptides in CHO cells, such as a Pcmv-Scriptvector (Stratagene, La Jolla, Calif.), pcDNA4 vectors (Invitrogen,Carlsbad, Calif.) and pCI-neo vectors (Promega, Madison, Wisc.). As willbe apparent, the subject gene constructs can be used to cause expressionof the subject ActRIIB polypeptides in cells propagated in culture,e.g., to produce proteins, including fusion proteins or variantproteins, for purification.

This invention also pertains to a host cell transfected with arecombinant gene including a coding sequence (e.g., SEQ ID NO: 4 or 6)for one or more of the subject ActRIIB polypeptide. The host cell may beany prokaryotic or eukaryotic cell. For example, an ActRIIB polypeptideof the invention may be expressed in bacterial cells such as E. coli,insect cells (e.g., using a baculovirus expression system), yeast, ormammalian cells. Other suitable host cells are known to those skilled inthe art.

Accordingly, the present invention further pertains to methods ofproducing the subject ActRIIB polypeptides. For example, a host celltransfected with an expression vector encoding an ActRIIB polypeptidecan be cultured under appropriate conditions to allow expression of theActRIIB polypeptide to occur. The ActRIIB polypeptide may be secretedand isolated from a mixture of cells and medium containing the ActRIIBpolypeptide. Alternatively, the ActRIIB polypeptide may be retainedcytoplasmically or in a membrane fraction and the cells harvested, lysedand the protein isolated. A cell culture includes host cells, media andother byproducts. Suitable media for cell culture are well known in theart. The subject ActRIIB polypeptides can be isolated from cell culturemedium, host cells, or both, using techniques known in the art forpurifying proteins, including ion-exchange chromatography, gelfiltration chromatography, ultrafiltration, electrophoresis, andimmunoaffinity purification with antibodies specific for particularepitopes of the ActRIIB polypeptides. In a preferred embodiment, theActRIIB polypeptide is a fusion protein containing a domain whichfacilitates its purification.

In another embodiment, a fusion gene coding for a purification leadersequence, such as a poly-(His)/enterokinase cleavage site sequence atthe N-terminus of the desired portion of the recombinant ActRIIBpolypeptide, can allow purification of the expressed fusion protein byaffinity chromatography using a Ni²⁺ metal resin. The purificationleader sequence can then be subsequently removed by treatment withenterokinase to provide the purified ActRIIB polypeptide (e.g., seeHochuli et al., (1987) J. Chromatography 411:177; and Janknecht et al.,PNAS USA 88:8972).

Techniques for making fusion genes are well known. Essentially, thejoining of various DNA fragments coding for different polypeptidesequences is performed in accordance with conventional techniques,employing blunt-ended or stagger-ended termini for ligation, restrictionenzyme digestion to provide for appropriate termini, filling-in ofcohesive ends as appropriate, alkaline phosphatase treatment to avoidundesirable joining, and enzymatic ligation. In another embodiment, thefusion gene can be synthesized by conventional techniques includingautomated DNA synthesizers. Alternatively, PCR amplification of genefragments can be carried out using anchor primers which give rise tocomplementary overhangs between two consecutive gene fragments which cansubsequently be annealed to generate a chimeric gene sequence (see, forexample, Current Protocols in Molecular Biology, eds. Ausubel et al.,John Wiley & Sons: 1992).

5. Exemplary Therapeutic Uses

In certain embodiments, compositions (e.g., ActRIIB polypeptides) of thepresent invention can be used for treating or preventing a disease orcondition that is associated with abnormal activity of an ActRIIBpolypeptide and/or an ActRIIB ligand (e.g., GDF8). These diseases,disorders or conditions are generally referred to herein as“ActRIIB-associated conditions.” In certain embodiments, the presentinvention provides methods of treating or preventing an individual inneed thereof through administering to the individual a therapeuticallyeffective amount of an ActRIIB polypeptide as described above. Thesemethods are particularly aimed at therapeutic and prophylactictreatments of animals, and more particularly, humans.

As used herein, a therapeutic that “prevents” a disorder or conditionrefers to a compound that, in a statistical sample, reduces theoccurrence of the disorder or condition in the treated sample relativeto an untreated control sample, or delays the onset or reduces theseverity of one or more symptoms of the disorder or condition relativeto the untreated control sample. The term “treating” as used hereinincludes prophylaxis of the named condition or amelioration orelimination of the condition once it has been established.

ActRIIB/ActRIIB ligand complexes play essential roles in tissue growthas well as early developmental processes such as the correct formationof various structures or in one or more post-developmental capacitiesincluding sexual development, pituitary hormone production, and creationof bone and cartilage. Thus, ActRIIB-associated conditions includeabnormal tissue growth and developmental defects. In addition,ActRIIB-associated conditions include, but are not limited to, disordersof cell growth and differentiation such as inflammation, allergy,autoimmune diseases, infectious diseases, and tumors.

Exemplary conditions for treatment include neuromuscular disorders(e.g., muscular dystrophy and muscle atrophy), congestive obstructivepulmonary disease (and muscle wasting associated with COPD), musclewasting syndrome, sarcopenia, cachexia, adipose tissue disorders (e.g.,obesity), type 2 diabetes, and bone degenerative disease (e.g.,osteoporosis). Other exemplary conditions include musculodegenerativeand neuromuscular disorders, tissue repair (e.g., wound healing),neurodegenerative diseases (e.g., amyotrophic lateral sclerosis),immunologic disorders (e.g., disorders related to abnormal proliferationor function of lymphocytes), and obesity or disorders related toabnormal proliferation of adipocytes.

In certain embodiments, compositions (e.g., ActRIIB-Fc polypeptides) ofthe invention are used as part of a treatment for a muscular dystrophy.The term “muscular dystrophy” refers to a group of degenerative musclediseases characterized by gradual weakening and deterioration ofskeletal muscles and sometimes the heart and respiratory muscles.Muscular dystrophies are genetic disorders characterized by progressivemuscle wasting and weakness that begin with microscopic changes in themuscle. As muscles degenerate over time, the person's muscle strengthdeclines. Exemplary muscular dystrophies that can be treated with aregimen including the subject ActRIIB polypeptides include: DuchenneMuscular Dystrophy (DMD), Becker Muscular Dystrophy (BMD),Emery-Dreifuss Muscular Dystrophy (EDMD), Limb-Girdle Muscular Dystrophy(LGMD), Facioscapulohumeral Muscular Dystrophy (FSH or FSHD) (also knownas Landouzy-Dejerine), Myotonic Dystrophy (MMD) (also known asSteinert's Disease), Oculopharyngeal Muscular Dystrophy (OPMD), DistalMuscular Dystrophy (DD), Congenital Muscular Dystrophy (CMD).

Duchenne Muscular Dystrophy (DMD) was first described by the Frenchneurologist Guillaume Benjamin Amand Duchenne in the 1860s. BeckerMuscular Dystrophy (BMD) is named after the German doctor Peter EmilBecker, who first described this variant of DMD in the 1950s. DMD is oneof the most frequent inherited diseases in males, affecting one in 3,500boys. DMD occurs when the dystrophin gene, located on the short arm ofthe X chromosome, is broken. Since males only carry one copy of the Xchromosome, they only have one copy of the dystrophin gene. Without thedystrophin protein, muscle is easily damaged during cycles ofcontraction and relaxation. While early in the disease musclecompensates by regeneration, later on muscle progenitor cells cannotkeep up with the ongoing damage and healthy muscle is replaced bynon-functional fibro-fatty tissue.

BMD results from different mutations in the dystrophin gene. BMDpatients have some dystrophin, but it is either insufficient in quantityor poor in quality. Having some dystrophin protects the muscles of thosewith BMD from degenerating as badly or as quickly as those of peoplewith DMD.

For example, recent researches demonstrate that blocking or eliminatingfunction of GDF8 (an ActRIIB ligand) in vivo can effectively treat atleast certain symptoms in DMD and BMD patients. Thus, the subjectActRIIB polypeptides may act as GDF8 inhibitors (antagonists), andconstitute an alternative means of blocking the functions of GDF8 and/orActRIIB in vivo in DMD and BMD patients. This approach is confirmed andsupported by the data shown herein, whereby an ActRIIB-Fc protein wasshown to increase muscle mass in a mouse model of muscular dystrophy.

Similarly, the subject ActRIIB polypeptides provide an effective meansto increase muscle mass in other disease conditions that are in need ofmuscle growth. For example, ALS, also called Lou Gehrig's disease (motorneuron disease) is a chronic, incurable, and unstoppable CNS disorderthat attacks the motor neurons, components of the CNS that connect thebrain to the skeletal muscles. In ALS, the motor neurons deteriorate andeventually die, and though a person's brain normally remains fullyfunctioning and alert, the command to move never reaches the muscles.Most people who get ALS are between 40 and 70 years old. The first motorneurons that weaken are those leading to the arms or legs. Those withALS may have trouble walking, they may drop things, fall slur theirspeech, and laugh or cry uncontrollably. Eventually the muscles in thelimbs begin to atrophy from disuse. This muscle weakness will becomedebilitating and a person will need a wheel chair or become unable tofunction out of bed. Most ALS patients die from respiratory failure orfrom complications of ventilator assistance like pneumonia, 3-5 yearsfrom disease onset. This approach is confirmed and supported by the datashown herein, whereby an ActRIIB-Fc protein was shown to improve theappearance, muscle mass and lifespan of a mouse model of ALS.

ActRIIB polypeptide-induced increased muscle mass might also benefitthose suffering from muscle wasting diseases. Gonzalez-Cadavid et al.(supra) reported that that GDF8 expression correlates inversely withfat-free mass in humans and that increased expression of the GDF8 geneis associated with weight loss in men with AIDS wasting syndrome. Byinhibiting the function of GDF8 in AIDS patients, at least certainsymptoms of AIDS may be alleviated, if not completely eliminated, thussignificantly improving quality of life in AIDS patients.

Since loss of GDF8 (an ActRIIB ligand) function is also associated withfat loss without diminution of nutrient intake (Zimmers et al., supra;McPherron and Lee, supra), the subject ActRIIB polypeptides may furtherbe used as a therapeutic agent for slowing or preventing the developmentof obesity and type II diabetes. This approach is confirmed andsupported by the data shown herein, whereby an ActRIIB-Fc protein wasshown to improve metabolic status in obese mice.

In certain embodiments, compositions (e.g., ActRIIB polypeptides) of theinvention are used as part of a treatment for metabolic syndrome (alsoknown as syndrome X and insulin resistance syndrome), which is acombination of disorders and risk factors that increase the risk ofdeveloping cardiovascular disease and diabetes mellitus type II. Mostpatients are older, obese, sedentary, and have some degree of insulinresistance. Central (abdominal or visceral) adiposity is a significantfeature of the syndrome.

In related embodiments, ActRIIB polypeptides and other compositions ofthe invention can be used as part of a treatment for diabetes mellitustype II (also known as non-insulin-dependent diabetes mellitus oradult-onset diabetes), which is characterized by elevated blood glucosein the context of insulin resistance and relative insulin deficiency.Complex and multifactorial metabolic changes in diabetes often lead todamage and functional impairment of many organs, most importantly thecardiovascular system. Diabetes mellitus type II is often associatedwith obesity (abdominal or visceral adiposity), hypertension, elevatedcholesterol, and metabolic syndrome. Important risk factors for diabetesmellitus type II include aging, high-fat diets, and a sedentarylifestyle.

In other related embodiments, ActRIIB polypeptides and othercompositions of the invention can be used as part of a treatment foratherosclerosis, a chronic inflammatory condition in which artery wallsthicken due to the accumulation of fatty deposits, often referred to asplaques. Risk factors for atherosclerosis include aging, diabetesmellitus, dyslipoproteinemia, obesity (abdominal or visceral adiposity),and a sedentary lifestyle.

ActRIIB polypeptides can also be used for lipodystrophic disorders,which tend to be associated with metabolic syndrome. Severe insulinresistance can result from both genetic and acquired forms oflipodystrophy, including in the latter case human immunodeficiency virus(HIV)-related lipodystrophy in patients treated with antiretroviraltherapy.

The cancer anorexia-cachexia syndrome is among the most debilitating andlife-threatening aspects of cancer. Progressive weight loss in canceranorexia-cachexia syndrome is a common feature of many types of cancerand is responsible not only for a poor quality of life and poor responseto chemotherapy, but also a shorter survival time than is found inpatients with comparable tumors without weight loss. Associated withanorexia, fat and muscle tissue wasting, psychological distress, and alower quality of life, cachexia arises from a complex interactionbetween the cancer and the host. It is one of the most common causes ofdeath among cancer patients and is present in 80% at death. It is acomplex example of metabolic chaos effecting protein, carbohydrate, andfat metabolism. Tumors produce both direct and indirect abnormalities,resulting in anorexia and weight loss. Currently, there is no treatmentto control or reverse the process. Cancer anorexia-cachexia syndromeaffects cytokine production, release of lipid-mobilizing andproteolysis-inducing factors, and alterations in intermediarymetabolism. Although anorexia is common, a decreased food intake aloneis unable to account for the changes in body composition seen in cancerpatients, and increasing nutrient intake is unable to reverse thewasting syndrome. Cachexia should be suspected in patients with cancerif an involuntary weight loss of greater than five percent of premorbidweight occurs within a six-month period.

Since systemic overexpression of GDF8 in adult mice was found to induceprofound muscle and fat loss analogous to that seen in human cachexiasyndromes (Zimmers et al., supra), the subject ActRIIB polypeptides aspharmaceutical compositions can be beneficially used to prevent, treat,or alleviate the symptoms of the cachexia syndrome, where muscle growthis desired.

In other embodiments, the present invention provides methods of inducingbone and/or cartilage formation, preventing bone loss, increasing bonemineralization or preventing the demineralization of bone. For example,the subject ActRIIB polypeptides and compounds identified in the presentinvention have application in treating osteoporosis and the healing ofbone fractures and cartilage defects in humans and other animals.ActRIIB polypeptides may be useful in patients that are diagnosed withsubclinical low bone density, as a protective measure against thedevelopment of osteoporosis.

In one specific embodiment, methods and compositions of the presentinvention may find medical utility in the healing of bone fractures andcartilage defects in humans and other animals. The subject methods andcompositions may also have prophylactic use in closed as well as openfracture reduction and also in the improved fixation of artificialjoints. De novo bone formation induced by an osteogenic agentcontributes to the repair of congenital, trauma-induced, or oncologicresection induced craniofacial defects, and also is useful in cosmeticplastic surgery. Further, methods and compositions of the invention maybe used in the treatment of periodontal disease, and in other toothrepair processes. In certain cases, the subject ActRIIB polypeptides mayprovide an environment to attract bone-forming cells, stimulate growthof bone-forming cells or induce differentiation of progenitors ofbone-forming cells. ActRIIB polypeptides of the invention may also beuseful in the treatment of osteoporosis. Further, ActRIIB polypeptidesmay be used in cartilage defect repair and prevention/reversal ofosteoarthritis.

In another specific embodiment, the invention provides a therapeuticmethod and composition for repairing fractures and other conditionsrelated to cartilage and/or bone defects or periodontal diseases. Theinvention further provides therapeutic methods and compositions forwound healing and tissue repair. The types of wounds include, but arenot limited to, burns, incisions and ulcers. See e.g., PCT PublicationNo. WO84/01106. Such compositions comprise a therapeutically effectiveamount of at least one of the ActRIIB polypeptides of the invention inadmixture with a pharmaceutically acceptable vehicle, carrier or matrix.

In another specific embodiment, methods and compositions of theinvention can be applied to conditions causing bone loss such asosteoporosis, hyperparathyroidism, Cushing's disease, thyrotoxicosis,chronic diarrheal state or malabsorption, renal tubular acidosis,chronic renal failure or anorexia nervosa. Many people know that beingfemale, having a low body weight, and leading a sedentary lifestyle arerisk factors for osteoporosis (loss of bone mineral density, leading tofracture risk). However, osteoporosis can also result from the long-termuse of certain medications. Osteoporosis resulting from drugs or anothermedical condition is known as secondary osteoporosis. In a conditionknown as Cushing's disease, the excess amount of cortisol produced bythe body results in osteoporosis and fractures. The most commonmedications associated with secondary osteoporosis are thecorticosteroids, a class of drugs that act like cortisol, a hormoneproduced naturally by the adrenal glands. Although adequate levels ofthyroid hormones (which are produced by the thyroid gland) are neededfor the development of the skeleton, excess thyroid hormone can decreasebone mass over time. Antacids that contain aluminum can lead to boneloss when taken in high doses by people with kidney problems,particularly those undergoing dialysis. Other medications that can causesecondary osteoporosis include phenytoin (Dilantin) and barbituratesthat are used to prevent seizures; methotrexate (Rheumatrex, Inmmunex,Folex PFS), a drug for some forms of arthritis, cancer, and immunedisorders; cyclosporine (Sandimmune, Neoral), a drug used to treat someautoimmune diseases and to suppress the immune system in organtransplant patients; luteinizing hormone-releasing hormone agonists(Lupron, Zoladex), used to treat prostate cancer and endometriosis;heparin (Calciparine, Liquaemin), an anticlotting medication; andcholestyramine (Questran) and colestipol (Colestid), used to treat highcholesterol. Gum disease causes bone loss because these harmful bacteriain our mouths force our bodies to defend against them. The bacteriaproduce toxins and enzymes under the gum-line, causing a chronicinfection.

In other embodiments, the present invention provides compositions andmethods for regulating body fat content in an animal and for treating orpreventing conditions related thereto, and particularly,health-compromising conditions related thereto. According to the presentinvention, to regulate (control) body weight can refer to reducing orincreasing body weight, reducing or increasing the rate of weight gain,or increasing or reducing the rate of weight loss, and also includesactively maintaining, or not significantly changing body weight (e.g.,against external or internal influences which may otherwise increase ordecrease body weight). One embodiment of the present invention relatesto regulating body weight by administering to an animal (e.g., a human)in need thereof an ActRIIB polypeptide.

In one specific embodiment, the present invention relates to methods andcompounds for reducing fat mass and/or reducing gain of fat mass in ananimal, and more particularly, for treating or ameliorating obesity inpatients at risk for or suffering from obesity. In another specificembodiment, the present invention is directed to methods and compoundsfor treating an animal that is unable to gain or retain weight (e.g., ananimal with a wasting syndrome). Such methods are effective to increasebody weight and/or mass, or to reduce weight and/or mass loss, or toimprove conditions associated with or caused by undesirably low (e.g.,unhealthy) body weight and/or mass.

7. Pharmaceutical Compositions

In certain embodiments, compounds (e.g., ActRIIB polypeptides) of thepresent invention are formulated with a pharmaceutically acceptablecarrier. For example, an ActRIIB polypeptide can be administered aloneor as a component of a pharmaceutical formulation (therapeuticcomposition). The subject compounds may be formulated for administrationin any convenient way for use in human or veterinary medicine.

In certain embodiments, the therapeutic method of the invention includesadministering the composition topically, systemically, or locally as animplant or device. When administered, the therapeutic composition foruse in this invention is, of course, in a pyrogen-free, physiologicallyacceptable form. Further, the composition may desirably be encapsulatedor injected in a viscous form for delivery to a target tissue site(e.g., bone, cartilage, muscle, fat or neurons), for example, a sitehaving a tissue damage. Topical administration may be suitable for woundhealing and tissue repair. Therapeutically useful agents other than theActRIIB polypeptides which may also optionally be included in thecomposition as described above, may alternatively or additionally, beadministered simultaneously or sequentially with the subject compounds(e.g., ActRIIB polypeptides) in the methods of the invention.

In certain embodiments, compositions of the present invention mayinclude a matrix capable of delivering one or more therapeutic compounds(e.g., ActRIIB polypeptides) to a target tissue site, providing astructure for the developing tissue and optimally capable of beingresorbed into the body. For example, the matrix may provide slow releaseof the ActRIIB polypeptides. Such matrices may be formed of materialspresently in use for other implanted medical applications.

The choice of matrix material is based on biocompatibility,biodegradability, mechanical properties, cosmetic appearance andinterface properties. The particular application of the subjectcompositions will define the appropriate formulation. Potential matricesfor the compositions may be biodegradable and chemically defined calciumsulfate, tricalciumphosphate, hydroxyapatite, polylactic acid andpolyanhydrides. Other potential materials are biodegradable andbiologically well defined, such as bone or dermal collagen. Furthermatrices are comprised of pure proteins or extracellular matrixcomponents. Other potential matrices are non-biodegradable andchemically defined, such as sintered hydroxyapatite, bioglass,aluminates, or other ceramics. Matrices may be comprised of combinationsof any of the above mentioned types of material, such as polylactic acidand hydroxyapatite or collagen and tricalciumphosphate. The bioceramicsmay be altered in composition, such as in calcium-aluminate-phosphateand processing to alter pore size, particle size, particle shape, andbiodegradability.

In certain embodiments, methods of the invention can be administered fororally, e.g., in the form of capsules, cachets, pills, tablets, lozenges(using a flavored basis, usually sucrose and acacia or tragacanth),powders, granules, or as a solution or a suspension in an aqueous ornon-aqueous liquid, or as an oil-in-water or water-in-oil liquidemulsion, or as an elixir or syrup, or as pastilles (using an inertbase, such as gelatin and glycerin, or sucrose and acacia) and/or asmouth washes and the like, each containing a predetermined amount of anagent as an active ingredient. An agent may also be administered as abolus, electuary or paste.

In solid dosage forms for oral administration (capsules, tablets, pills,dragees, powders, granules, and the like), one or more therapeuticcompounds of the present invention may be mixed with one or morepharmaceutically acceptable carriers, such as sodium citrate ordicalcium phosphate, and/or any of the following: (1) fillers orextenders, such as starches, lactose, sucrose, glucose, mannitol, and/orsilicic acid; (2) binders, such as, for example, carboxymethylcellulose,alginates, gelatin, polyvinyl pyrrolidone, sucrose, and/or acacia; (3)humectants, such as glycerol; (4) disintegrating agents, such asagar-agar, calcium carbonate, potato or tapioca starch, alginic acid,certain silicates, and sodium carbonate; (5) solution retarding agents,such as paraffin, (6) absorption accelerators, such as quaternaryammonium compounds; (7) wetting agents, such as, for example, cetylalcohol and glycerol monostearate; (8) absorbents, such as kaolin andbentonite clay; (9) lubricants, such a talc, calcium stearate, magnesiumstearate, solid polyethylene glycols, sodium lauryl sulfate, andmixtures thereof; and (10) coloring agents. In the case of capsules,tablets and pills, the pharmaceutical compositions may also comprisebuffering agents. Solid compositions of a similar type may also beemployed as fillers in soft and hard-filled gelatin capsules using suchexcipients as lactose or milk sugars, as well as high molecular weightpolyethylene glycols and the like.

Liquid dosage forms for oral administration include pharmaceuticallyacceptable emulsions, microemulsions, solutions, suspensions, syrups,and elixirs. In addition to the active ingredient, the liquid dosageforms may contain inert diluents commonly used in the art, such as wateror other solvents, solubilizing agents and emulsifiers, such as ethylalcohol, isopropyl alcohol, ethyl carbonate, ethyl acetate, benzylalcohol, benzyl benzoate, propylene glycol, 1,3-butylene glycol, oils(in particular, cottonseed, groundnut, corn, germ, olive, castor, andsesame oils), glycerol, tetrahydrofuryl alcohol, polyethylene glycolsand fatty acid esters of sorbitan, and mixtures thereof. Besides inertdiluents, the oral compositions can also include adjuvants such aswetting agents, emulsifying and suspending agents, sweetening,flavoring, coloring, perfuming, and preservative agents.

Suspensions, in addition to the active compounds, may contain suspendingagents such as ethoxylated isostearyl alcohols, polyoxyethylenesorbitol, and sorbitan esters, microcrystalline cellulose, aluminummetahydroxide, bentonite, agar-agar and tragacanth, and mixturesthereof.

Certain compositions disclosed herein may be administered topically,either to skin or to mucosal membranes. The topical formulations mayfurther include one or more of the wide variety of agents known to beeffective as skin or stratum comeum penetration enhancers. Examples ofthese are 2-pyrrolidone, N-methyl-2-pyrrolidone, dimethylacetamide,dimethylformamide, propylene glycol, methyl or isopropyl alcohol,dimethyl sulfoxide, and azone. Additional agents may further be includedto make the formulation cosmetically acceptable. Examples of these arefats, waxes, oils, dyes, fragrances, preservatives, stabilizers, andsurface active agents. Keratolytic agents such as those known in the artmay also be included. Examples are salicylic acid and sulfur.

Dosage forms for the topical or transdermal administration includepowders, sprays, ointments, pastes, creams, lotions, gels, solutions,patches, and inhalants. The active compound may be mixed under sterileconditions with a pharmaceutically acceptable carrier, and with anypreservatives, buffers, or propellants which may be required. Theointments, pastes, creams and gels may contain, in addition to a subjectcompound of the invention (e.g., an ActRIIB polypeptide), excipients,such as animal and vegetable fats, oils, waxes, paraffins, starch,tragacanth, cellulose derivatives, polyethylene glycols, silicones,bentonites, silicic acid, talc and zinc oxide, or mixtures thereof.

Powders and sprays can contain, in addition to a subject compound,excipients such as lactose, talc, silicic acid, aluminum hydroxide,calcium silicates, and polyamide powder, or mixtures of thesesubstances. Sprays can additionally contain customary propellants, suchas chlorofluorohydrocarbons and volatile unsubstituted hydrocarbons,such as butane and propane.

In certain embodiments, pharmaceutical compositions suitable forparenteral administration may comprise one or more ActRIIB polypeptidesin combination with one or more pharmaceutically acceptable sterileisotonic aqueous or nonaqueous solutions, dispersions, suspensions oremulsions, or sterile powders which may be reconstituted into sterileinjectable solutions or dispersions just prior to use, which may containantioxidants, buffers, bacteriostats, solutes which render theformulation isotonic with the blood of the intended recipient orsuspending or thickening agents. Examples of suitable aqueous andnonaqueous carriers which may be employed in the pharmaceuticalcompositions of the invention include water, ethanol, polyols (such asglycerol, propylene glycol, polyethylene glycol, and the like), andsuitable mixtures thereof vegetable oils, such as olive oil, andinjectable organic esters, such as ethyl oleate. Proper fluidity can bemaintained, for example, by the use of coating materials, such aslecithin, by the maintenance of the required particle size in the caseof dispersions, and by the use of surfactants.

The compositions of the invention may also contain adjuvants, such aspreservatives, wetting agents, emulsifying agents and dispersing agents.Prevention of the action of microorganisms may be ensured by theinclusion of various antibacterial and antifungal agents, for example,paraben, chlorobutanol phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption, such as aluminum monostearate andgelatin.

It is understood that the dosage regimen will be determined by theattending physician considering various factors which modify the actionof the subject compounds of the invention (e.g., ActRIIB polypeptides).The various factors will depend upon the disease to be treated.

In certain embodiments, the present invention also provides gene therapyfor the in vivo production of ActRIIB polypeptides or other compoundsdisclosed herein. Such therapy would achieve its therapeutic effect byintroduction of the ActRIIB polynucleotide sequences into cells ortissues having the disorders as listed above. Delivery of ActRIIBpolynucleotide sequences can be achieved using a recombinant expressionvector such as a chimeric virus or a colloidal dispersion system.Preferred for therapeutic delivery of ActRIIB polynucleotide sequencesis the use of targeted liposomes.

Various viral vectors which can be utilized for gene therapy as taughtherein include adenovirus, herpes virus, vaccinia, or, preferably, anRNA virus such as a retrovirus. Preferably, the retroviral vector is aderivative of a murine or avian retrovirus. Examples of retroviralvectors in which a single foreign gene can be inserted include, but arenot limited to: Moloney murine leukemia virus (MoMuLV), Harvey murinesarcoma virus (HaMuSV), murine mammary tumor virus (MuMTV), and RousSarcoma Virus (RSV). A number of additional retroviral vectors canincorporate multiple genes. All of these vectors can transfer orincorporate a gene for a selectable marker so that transduced cells canbe identified and generated. Retroviral vectors can be madetarget-specific by attaching, for example, a sugar, a glycolipid, or aprotein. Preferred targeting is accomplished by using an antibody. Thoseof skill in the art will recognize that specific polynucleotidesequences can be inserted into the retroviral genome or attached to aviral envelope to allow target specific delivery of the retroviralvector containing the ActRIIB polynucleotide. In one preferredembodiment, the vector is targeted to bone, cartilage, muscle or neuroncells/tissues.

Alternatively, tissue culture cells can be directly transfected withplasmids encoding the retroviral structural genes gag, pol and env, byconventional calcium phosphate transfection. These cells are thentransfected with the vector plasmid containing the genes of interest.The resulting cells release the retroviral vector into the culturemedium.

Another targeted delivery system for ActRIIB polynucleotides is acolloidal dispersion system. Colloidal dispersion systems includemacromolecule complexes, nanocapsules, microspheres, beads, andlipid-based systems including oil-in-water emulsions, micelles, mixedmicelles, and liposomes. The preferred colloidal system of thisinvention is a liposome. Liposomes are artificial membrane vesicleswhich are useful as delivery vehicles in vitro and in vivo. RNA, DNA andintact virions can be encapsulated within the aqueous interior and bedelivered to cells in a biologically active form (see e.g., Fraley, etal., Trends Biochem. Sci., 6:77, 1981). Methods for efficient genetransfer using a liposome vehicle, are known in the art, see e.g.,Mannino, et al., Biotechniques, 6:682, 1988. The composition of theliposome is usually a combination of phospholipids, usually incombination with steroids, especially cholesterol. Other phospholipidsor other lipids may also be used. The physical characteristics ofliposomes depend on pH, ionic strength, and the presence of divalentcations.

Examples of lipids useful in liposome production include phosphatidylcompounds, such as phosphatidylglycerol, phosphatidylcholine,phosphatidylserine, phosphatidylethanolamine, sphingolipids,cerebrosides, and gangliosides. Illustrative phospholipids include eggphosphatidylcholine, dipalmitoylphosphatidylcholine, anddistearoylphosphatidylcholine. The targeting of liposomes is alsopossible based on, for example, organ-specificity, cell-specificity, andorganelle-specificity and is known in the art.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain embodiments andembodiments of the present invention, and are not intended to limit theinvention.

Example 1 Generation of ActRIIB(25-131)-hFc with Alternative NucleotideSequences

To generate ActRIIB(25-131)-hFc, the human ActRIIB extracellular domainwith N-terminal and C-terminal truncations (residues 25-131 of thenative protein) was fused N-terminally with a TPA leader sequencesubstituted for the native ActRIIB leader and C-terminally with a humanFc domain via a minimal linker (three glycine residues) (FIG. 1). Anucleotide sequence encoding this fusion protein is shown in FIG. 2.Applicants modified the codons and found a variant nucleic acid encodingthe ActRIIB(25-131)-hFc protein that provided substantial improvement inthe expression levels of initial transformants (FIG. 3).

The mature protein has an amino acid sequence as follows (N-terminusconfirmed by N-terminal sequencing)(SEQ ID NO: 8):

ETRECIYYNA NWELERTNQS GLRECEGEQD KRLHCYASWRNSSGTIELVK KGCWLDDFNC YDRQECVATE ENPQVYFCCCEGNFCNERFT HLPEAGGPEV TYEPPPTGGG THTCPPCPAPELLGGPSVFL FPPKPKDTLM ISRTPEVTCV VVDVSHEDPEVKFNWYVDGV EVHNAKTKPR EEQYNSTYRV VSVLTVLHQDWLNGKEYKCK VSNKALPAPI EKTISKAKGQ PREVQVYTLPPSREEMTKNQ VSLTCLVKGF YPSDIAVEWE SNGQPENNYKTTPPVLDSDG SFFLYSKLTV DKSRWQQGNV FSCSVMHEAL HNHYTQKSLS LSPGKAmino acids 1-107 are derived from ActRIIB.

The expressed molecule was purified using a series of columnchromatography steps, including for example, three or more of thefollowing, in any order: Protein A chromatography, Q sepharosechromatography, phenylsepharose chromatography, size exclusionchromatography and cation exchange chromatography. The purificationcould be completed with viral filtration and buffer exchange.

Example 2 High-Affinity Ligand Binding by ActRIIB(25-131)-hFc

Affinities of several ligands for ActRIIB(25-131)-hFc and itsfill-length counterpart ActRIIB(20-134)-hFc were evaluated in vitro witha Biacore™ instrument, and the results are summarized in the tablebelow. Kd values were obtained by steady-state affinity fit due to veryrapid association and dissociation of the complex, which preventedaccurate determination of k_(on) and k_(off). ActRIIB(25-131)-hFc boundactivin A, activin B, and GDF11 with high affinity. Intriguingly,ActRIIB(25-131)-hFc appears to show a higher affinity for GDF3 thanActRIIB(20-134)-hFc (data not shown).

Ligand Affinities of ActRIIB-hFc Forms:

Activin A Activin B GDF11 Fusion Construct (e-11) (e-11) (e-11)ActRIIB(20-134)-hFc 1.6 1.2 3.6 ActRIIB(25-131)-hFc 1.8 1.2 3.1

Example 3 ActRIIB(25-131)-hFc Increases Muscle Mass and Strength In Vivo

Applicants investigated the ability of ActRIIB(25-131)-hFc to increasemuscle mass and strength in the mouse. Male mice (n=10 per group) weretreated subcutaneously twice per week with vehicle (Tris-bufferedsaline) or one of five doses of ActRIIB(25-131)-hFc. Four weeks oftreatment with ActRIIB(25-131)-hFc produced a clear dose-dependentincrease in lean tissue mass (FIG. 4), as determined by whole-bodynuclear magnetic resonance (NMR) scanning. Increased muscle mass wasconfirmed at study termination for specific muscles, including thepectoralis (FIG. 5), rectus femoris, and gastrocnemius. Importantly,increased muscle mass was accompanied by increased strength, as assessedby grip strength, compared to vehicle (FIG. 6). These results providecompelling evidence that ActRIIB(25-13)-hFc increases both muscle massand muscle strength in vivo.

Example 4 ActRIIB(25-131)-hFc Prevents Muscle Loss in Mouse Model ofAndrogen Deprivation

Applicants investigated the ability of ActRIIB(25-131)-hFc to preventmuscle loss in a mouse model of androgen deprivation, a standardtherapeutic intervention for advanced prostate cancer in men. Male mice(n=10 per group) were orchidectomized (ORX) or sham-operated and treatedsubcutaneously twice per week with TBS vehicle, ActRIIB(25-13)-hFc at 10mg/kg, or its full-length murine counterpart ActRIIB(20-134)-mFc at 10mg/kg. Lean tissue mass was determined by whole-body NMR scan. ORX micetreated for four weeks with either of the ActRIIB-Fc forms displayed anincrease in lean tissue mass from baseline, which was highly significantcompared to the decrease observed in ORX controls over that period (FIG.7). An analogous, highly significant increase was observed undergonad-intact conditions for both ActRIIB-Fc forms compared to shamcontrols (FIG. 7). These results demonstrate that ActRIIB(25-131)-hFccan increase lean tissue mass (prevent muscle loss) as effectively asits full-length counterpart ActRIIB(20-134)-mFc in this androgendeprivation model.

Example 5 ActRIIB(25-131)-hFc Improves Body Composition in Mouse Modelof Diet-Induced Obesity

Applicants also investigated the ability of ActRIIB(25-131)-hFc toincrease muscle mass and reduce fat mass in a mouse model ofdiet-induced obesity. Male mice (n=10 per group) were fed either astandard chow diet or a high fat diet and treated intraperitoneallytwice per week with TBS vehicle or ActRIIB(25-131)-hFc at 10 mg/kg. Leantissue mass and fat mass were determined by whole-body NMR scan.Treatment of mice on the high fat diet with ActRIIB(25-131)-hFc for fourweeks resulted in more than a 25% increase in lean tissue mass ascompared to a 2% increase with vehicle treatment (FIG. 8). Similarresults were obtained in mice on the control diet withActRIIB(25-131)-hFc as compared to vehicle (FIG. 8). Moreover, continuedtreatment was found to improve adiposity. Compared to vehicle,ActRIIB(25-131)-hFc treatment for 12 weeks reduced fat mass byapproximately half in mice on the high fat diet as well as in those onthe control diet (FIG. 9).

Taken together, these data demonstrate that ActRIIB(25-131)-hFc can beused to improve body composition in vivo under a variety of conditions,including androgen deprivation and high fat intake.

Example 6 ActRIIB(25-131)-hFc Normalizes Serum Lipids, Insulin, andAdiponectin in Mouse Model of Diet-Induced Obesity

Applicants investigated the effects of ActRIIB(25-131)-hFc on serumconcentrations of clinically important lipids, insulin, adiponectin, andon other metabolic endpoints in male mice fed a high-fat diet.Ten-week-old C57BL/6 mice were weight-matched and treated withActRIIB(25-131)-hFc (n=10) or Tris-buffered-saline (TBS) vehicle (n=7)twice per week at 10 mg/kg, s.c., for 60 days. During this period, micehad unlimited access to a diet containing 58% fat instead of thestandard chow containing 4.5% fat.

ActRIIB(25-131)-hFc treatment caused a constellation of noteworthymetabolic effects. In mice fed a high-fat diet, ActRIIB(25-131)-hFcreduced the pathologically elevated serum concentrations oftriglycerides, free fatty acids, high-density lipoprotein (HDL), andlow-density lipoprotein (LDL) (FIG. 10-13), in most cases normalizingthese parameters to levels observed in mice fed a standard diet.Importantly, ActRIIB(25-131)-hFc treatment also normalized insulinconcentrations in high-fat-diet mice (FIG. 14) and increasedconcentrations of adiponectin significantly above even those in mice feda standard diet (FIG. 15). Adiponectin is a key biomarker of bodycomposition, as circulating adiponectin levels are known to varyinversely with fat mass/obesity, and adiponectin enhances insulinsensitivity in target tissues. ActRIIB(25-131)-hFc also reduced serumconcentrations of leptin, another major indicator of adipocyte status,by nearly 50% (P<0.05). Finally, the aforementioned effects wereaccompanied by beneficial changes in body composition, as determined bynuclear magnetic resonance (NMR) at baseline and Day 48. Under high-fatdietary conditions, total fat mass in vehicle-treated controls tripledduring this 48-day period, and ActRIIB(25-131)-hFc treatment cut thisincrease by nearly 40%. By Day 48, total fat mass was 27% of body weightin ActRIIB-Fc-treated mice vs. 39% in control mice, whereas lean tissuemass was 59% of body weight in ActRIIB(25-131)-hFc-treated mice vs. 55%in control mice. Thus, the net result was a healthier body compositionunder conditions of high-fat diet.

For the foregoing serum parameters, ActRIIB(25-131)-hFc consistentlyoutperformed ActRIIB(20-134)-hFc, which was also evaluated in this samestudy. Thus, ActRIIB(25-131)-hFc improved triglyceride levels nearly 6times as much, FFA levels nearly twice as much, HDL levels nearly 4times as much, insulin levels more than twice as much, and adiponectinlevels nearly 1.5 times as much as ActRIIB(20-134)-hFc did at the samedose.

Example 7 ActRIIB(25-131)-hFc Induces Thermogenic Properties in WhiteFat in Mouse Model of Diet-Induced Obesity

In the study described above (Example 6), Applicants also investigatedeffects of ActRIIB(25-131)-hFc on thermogenic properties of whiteadipose tissue. Under high-fat dietary conditions, ActRIIB(25-131)-hFctreatment triggered histological changes and a gene expression profilein white adipose tissue that were consistent with thermogeniccapability. As shown in FIG. 16, histological examination of epididymalwhite fat indicated that ActRIIB(25-131)-hFc reduced lipid droplet sizeand caused formation of clusters of multilocular adipocytes that are ahallmark of brown fat. Moreover, immunohistochemical analysis of thistissue revealed widespread cytoplasmic induction of UCP1 in bothmultilocular and unilocular adipocytes as a result ofActRIIB(25-131)-hFc treatment (FIG. 16).

Accompanying these histological changes were significant changes in theexpression of key thermogenic and metabolic regulatory genes inepididymal white fat, as determined by quantitative RT-PCR (reversetranscription polymerase chain reaction). In mice on the high-fat diet.ActRIIB(25-131)-hFc treatment increased UCP1 mRNA levels more than60-fold compared to vehicle (FIG. 17), a particularly impressive changesince this strain of mouse displays severely blunted induction of UCP1and brown adipocytes within key white fat depots compared to other mousestrains (Guerra et al., 1998, J Clin Invest 102:412-420; Xue et al.,2007, J Lipid Res 48:41-51). In addition, ActRIIB(25-131)-hFc treatmentincreased levels of mRNA encoding the sirtuin SIRT-1 (silent informationregulator two, homolog 1), an energy-sensitive master regulator(deacetylase) that protects against metabolic damage induced by ahigh-fat diet (Pfluger et al, 2008, Proc Natl Acad Sci USA105:9793-9798) and is implicated as an important control of fatty acidmobilization (Rodgers et al., 2008, FEBS Lett 582:46-53). Significantly,ActRIIB(25-131)-hFc treatment also increased levels of mRNA encodingPGC-1α (peroxisome proliferator-activated receptor gammacoactivator-1α), a well-documented target of SIRT-1 that, in turn,controls expression of many genes necessary for mitochondrial biogenesisand thermogenic capability in brown adiopose tissue (Uldry et al., 2006,Cell Metab, 3:333-341). Notably, forced expression of PGC-1α in whiteadipocytes has been shown to induce a thermogenic program of geneexpression, including UCP1, closely resembling that in brown adipocytes(Hansen et al., 2006, Biochem J 398:153-168). In the present study,ActRIIB(25-13)-hFc restored PGC-1α gene expression in white adiposetissue under high-fat dietary conditions to levels indistinguishablefrom those in mice fed the standard diet.

Additional changes associated with treatment constitute a prominent linkbetween the altered expression profile in white adipose tissue andbeneficial hormonal and metabolic effects. Thus, in epididymal whitefat, ActRIIB(25-131)-hFc increased levels of mRNA encoding Foxo-1(forkhead box-containing, protein O subfamily-1), a transcription factorthat is both a target of SIRT-1 and a key inducer of adiponectinexpression (Qiao et al., 2006, J Biol Chem 281:39915-39924). Consistentwith Foxo-1 mRNA induction, ActRIIB(25-131)-hFc treatment raised levelsof adiponectin mRNA in white fat (FIG. 18), which helps to account forincreased circulating levels of adiponectin (FIG. 15, Example 6),enhanced insulin sensitivity in target tissues, and normalized insulinconcentrations (FIG. 14, Example 6) in these animals. In summary,ActRIIB(25-131)-hFc treatment under high-fat dietary conditions resultedin 1) histological changes and a gene expression profile in whiteadipose tissue that were consistent with thermogenic capability and 2)beneficial changes in a wide range of hormonal and metabolic parameters.

Example 8 Effects of ActRIIB(25-131)-hFc on Liver and Muscle in MouseModel of Diet-Induced Obesity

Nonalcoholic fatty liver disease (NAFLD) is a spectrum of increasinglycommon hepatic disorders widely considered to be the hepaticmanifestation of metabolic syndrome and characterized by fataccumulation in the liver (steatosis), often with deleterious effects. Asubset of NAFLD patients develop an inflammatory condition referred toas nonalcoholic steatohepatitis (NASH), which can progress further tohepatic fibrosis, cirrhosis, and hepatocellular carcinoma (Perlemuter etal, 2007, Nat Clin Pract Endocrinol Metab 3:458-469). In the studydescribed above (Examples 6-7), Applicants investigated whetherActRIIB(25-131)-hFc could inhibit hepatic steatosis associated with ahigh-fat diet. At study completion, hepatic tissue of mice fed thehigh-fat diet displayed large numbers of densely packed lipid droplets,as assessed by staining with Oil Red O, whereas mice fed the standarddiet showed no evidence of hepatic lipid deposits (FIG. 19). Treatmentwith ActRIIB(25-131)-hFc almost completely reversed hepatic lipiddeposition and normalized the appearance of hepatic tissue despite thehigh-fat diet. Thus, ActRIIB(25-131)-hFc was an effective inhibitor ofhepatic steatosis caused by high-fat diet.

ActRIIB(25-131)-hFc treatment also increased muscle mass in this modelof diet-induced obesity, consistent with findings in other models(Examples 3-5). Specifically, ActRIIB(25-131)-hFc increased pectoralismass by more than 70% (P<0.001), gastrocnemius mass by nearly 40%(P<0.001), and rectus femoris mass by more than 25% (P<0.001) comparedto high-fat diet controls. These changes in muscle mass were accompaniedby changes in muscle gene expression, as determined in gastrocnemiustissue by RT-PCR. Compared to high-fat diet controls,ActRIIB(25-131)-hFc increased PGC-1α mRNA levels and Foxo-1 mRNA levelsby approximately 50% each (P<0.05) in gastrocnemius.

Example 9 Effect of ActRIIB(25-131)-mFc on Visceral White Fat in MouseModel of Diet-Induced Obesity

Accumulation of visceral fat, as opposed to subcutaneous fat, plays acritical role in the development of cardiovascular disease andobesity-related disorders such as diabetes mellitus, hyperlipidemia,hypertension, and metabolic syndrome (Matsuzawa et al., 2006, FEBS Lett580:2917-2921). Due to its location, visceral (or intra-abdominal) fathas ready access to the liver via the hepatic portal circulation, whereit could influence metabolism, promote insulin resistance, and causesteatosis. Therefore, in a study similar to that described above(Examples 6-8), Applicants investigated effects of the truncated variantActRIIB(25-131)-mFc on the quantities of visceral fat vs. abdominalsubcutaneous fat under high-fat dietary conditions. Nine-week-oldC57BL/6 mice were treated with ActRIIB(25-131)-mFc (n=20), at 10 mg/kg,s.c., or Tris-buffered-saline (TBS) vehicle (n=10) twice per week for 60days. Beginning 7 days before the start of dosing, mice had unlimitedaccess to a diet containing 58% fat instead of the standard chowcontaining 4.5% fat. An additional group of mice (n=10) maintained onthe standard chow diet was also treated with TBS vehicle and followed asa dietary control. Fat volumes were determined by microCT for a subsetof mice (n=4 per group) whose percentages of total body fat, asdetermined by nuclear magnetic resonance (NMR) analysis, were closest togroup means (all mice were subjected to NMR analysis).

Visceral fat and abdominal subcutaneous fat both varied markedly in sizewith diet and ActRIIB(25-131)-mFc treatment. Three-dimensionalreconstruction of microCT images obtained partway through the study (35days) demonstrates that the depots of visceral fat and subcutaneous fatboth expanded as a result of the high-fat diet and thatActRIIB(25-131)-mFc largely reversed those increases (FIG. 20). Whenanalyzed quantitatively at study conclusion (60 days), the effect ofActRIIB(25-131)-mFc compared to high-fat diet alone was highlysignificant for both visceral fat (FIG. 21) and abdominal subcutaneousfat (FIG. 22).

Example 10 Effect of ActRIIB(25-131)-mFc on Brown Fat Properties inMouse Model of Diet-Induced Obesity

In the study described in Example 9, Applicants also investigatedeffects of ActRIIB(25-131)-mFc on properties of intrascapular brown fatdepots under high-fat dietary conditions. Compared to the standard diet,the high-fat diet produced several changes in the interscapular depot ofbrown adipose tissue, and ActRIIB(25-131)-mFc treatment eithercompletely or largely reversed each of these changes. Specifically,high-fat diet caused a pronounced enlargement of the interscapular depotas well as lightening of its color from red to pink (FIG. 23). Thisdiet-induced enlargement reflected a doubling of the mass (FIG. 24) anda reduction in the density (FIG. 25) of brown fat depots. Depot densitywas determined by micro-computed tomography (microCT) in situ for asubset of mice (n=4 per group) whose percentages of total body fat, asdetermined by nuclear magnetic resonance (NMR) analysis, were closest tothe group means (all mice were subjected to NMR analysis).ActRIIB(25-131)-mFc treatment completely reversed diet-induced changesin brown fat mass (FIG. 24) and density (FIG. 25), while largelyreversing diet-induced changes in size and color of the depot (FIG. 23).These results indicate that, under high-fat dietary conditions,ActRIIB(25-131)-mFc largely or completely restores properties likely tocorrelate with healthy brown fat function and thus improves the qualityof brown fat as it decreases the overall size of brown fat depots.

Example 11 Effects of ActRIIB(25-131)-mFc on Muscle, Bone, Fat, andMetabolic Hormones in Mouse Model of Aging

Body composition changes with aging in a predictable manner. Normalage-dependent decline in muscle mass and strength, known as sarcopenia,begins around age 30 and accelerates after age 60 (Stenholm et al, 2008,Curr Opin Clin Nutr Metab Care 11:693-700). Bone mass and strengthexhibit a similar decline with age, leading to an increased risk ofosteoporosis in the elderly. Whole-body fat mass increases with ageuntil around age 70, then declines in absolute terms but remains aroughly constant proportion of total body mass (Cartwright et al., 2007,Exp Gerontol 42:463-471). Based on efficacy observed in other models anddescribed herein, Applicants investigated effects of ActRIIB(25-131)-mFcon muscle, bone, fat, and insulin levels in a mouse model of aging.Nineteen-month-old male C57BL/6 mice were given unlimited access to astandard chow diet and treated with ActRIIB(25-131)-mFc (n=16), at 10mg/kg, s.c., or TBS vehicle (n=15) twice per week for 8 weeks. As aframe of reference, median life expectancy in this mouse strain waspreviously found to be approximately 27 months under standard dietaryconditions (Turturro et al., 2002, J Gerontol A Biol Sci Med Sci57:B379-389).

ActRIIB(25-131)-mFc treatment generated a series of notable changes inbody composition and metabolic hormone effects in these aged mice. Asdetermined by whole-body NMR analysis, lean tissue mass was essentiallyunchanged in control mice over the course of the study, whereas inActRIIB(24-131)-mFc-treated mice it increased progressively to almost20% above baseline by 7 weeks (FIG. 26). Consistent with this whole-bodyeffect, ActRIIB(25-131)-mFc also significantly increased the mass ofindividual muscle groups, including the pectoralis (increased 55%),rectus femoris (40%), triceps (40%), and gastrocnemius (28%), comparedto vehicle-treated controls at 8 weeks. Importantly, ActRIIB(25-131)-mFctreatment improved neuromuscular function, as determined by forelimbgrip strength testing according to an established protocol(http://jaxservices.jax.org/phenotyping/gripstrength_protocol.html)(FIG. 27).

Several bone-related parameters improved with ActRIIB(25-131)-mFctreatment in aged mice. As determined by DEXA analysis at baseline and8-week time points, ActRIIB(25-131)-mFc increased whole-body bonemineral density over the course of the study, whereas controls wereessentially unchanged (FIG. 28). In addition, microCT analysis of theproximal tibia demonstrated that ActRIIB(25-131)-mFc treatment for 8weeks doubled the bone volume fraction of the proximal tibia compared tocontrols (P<0.01).

ActRIIB(25-131)-mFc exerted major effects on fat in aged mice. Asdetermined by NMR analysis at multiple time points, there was aprogressive decline in whole-body fat mass in vehicle-treated controlsover the course of the study (FIG. 29), consistent with findings fromhumans in advanced old age. ActRIIB(25-131)-mFc treatment acceleratedthis change, triggering a decrease of twice the magnitude observed incontrols (−44% vs. −19%, respectively) (FIG. 29). By the terminal timepoint, ActRIIB(25-131)-mFc significantly reduced the mass of theindividual epididymal, inguinal, and retroperitoneal depots of white fatby amounts ranging from 48-54%. Interestingly, ActRIIB(25-131)-mFctreatment also reduced the mass of the interscapular brown fat depots bynearly 45% (P<0.05), similar to results obtained for this tissue in themouse model of dietary obesity (Example 10). Finally, as determined bymicroCT analysis in a representative subset of mice (n=4) from eachgroup, ActRIIB(25-131)-mFc reduced the volume of the visceral componentof abdominal fat by 65% (P<0.01) and the subcutaneous component ofabdominal fat by 49% (P<0.01). Hence, the critical visceral fatcompartment was strongly targeted by ActRIIB(25-131)-mFc in this modelof aging.

ActRIIB(25-131)-mFc also produced beneficial changes in importantmetabolic hormones in aged mice. Eight weeks of treatment withActRIIB(25-131)-mFc nearly doubled circulating adiponectinconcentrations (P<0.001) and reduced circulating insulin concentrationsby more than 40% (FIG. 30). An elevated fasting insulin concentration(hyperinsulinemia) is a widely accepted surrogate measure of insulinresistance (Weyer et al., 2000, Diabetes 49:2094-2101), and increasedadiponectin concentrations are likely contributing to improved insulinsensitivity in the present study. Glycated hemoglobin (A1C)concentrations were significantly reduced by ActRIIB(25-131)-mFc in thisstudy (FIG. 31), thereby providing additional evidence for improvedglucose regulation with ActRIIB(25-131)-mFc treatment in this model ofaging.

Example 12 Effect of ActRIIB(25-131)-hFc on Lean Tissue in Mouse Modelof Cancer Cachexia

Cachexia is undesired weight loss resulting from loss of muscle andadipose tissue. Many tumors are associated with loss of appetite andsevere muscle loss, and patients exhibiting cachexia have a poorerprognosis than non-cachectic patients. Since the colon-cancer cell lineCT26 induces profound cachexia in mice, ActRIIB(25-131)-hFc was testedin this mouse model for potential effects on xenograft-induced cachexia.Eight-week-old BALB/c mice were injected subcutaneously with 10⁶Colon-26 adenocarcinoma (CT26) cells per mouse. Two weeks after tumorimplantation, treatment was initiated with ActRIIB(25-131)-hFc (n=15),at 10 mg/kg, s.c., or Tris-buffered-saline (TBS) vehicle (n=13) twiceper week. Additional groups of BALB/c mice did not receive CT26 cellsbut were treated with ActRIIB(25-131)-hFc or vehicle as above. Treatmentwith ActRIIB(25-131)-hFc resulted in a significant increase in bodyweight that was maintained across the study. At 5 weeks post tumorimplantation, vehicle-treated mice exhibited a 7% loss of lean tissuemass from baseline, as determined by NMR analysis, whereas mice treatedwith ActRIIB(25-131)-hFc exhibited a 27% increase in lean mass frombaseline (FIG. 32). Fat mass did not differ significantly between thegroups. These results demonstrate that ActRIIB(25-131)-hFc can alleviatecachexia in tumor-bearing mice and could be an effective therapy fortreating cachexia in cancer patients.

Taken together, these data indicate that ActRIIB(25-131)-hFc fusionprotein can be used as an antagonist of signaling by TGF-family ligandsto reverse many pathological metabolic changes associated withdiet-induced obesity, and thereby, to treat metabolic conditionsexacerbated by high caloric intake. Moreover, ActRIIB(25-131)-hFc can beused to treat pathologic metabolic changes associated with aging orcancer cachexia.

INCORPORATION BY REFERENCE

All publications and patents mentioned herein are hereby incorporated byreference in their entirety as if each individual publication or patentwas specifically and individually indicated to be incorporated byreference.

While specific embodiments of the subject matter have been discussed,the above specification is illustrative and not restrictive. Manyvariations will become apparent to those skilled in the art upon reviewof this specification and the claims below. The full scope of theinvention should be determined by reference to the claims, along withtheir full scope of equivalents, and the specification, along with suchvariations.

We claim:
 1. A method for producing an ActRIIB-Fc fusion polypeptide,comprising: a) culturing a host cell under conditions suitable forexpression of the polypeptide, and b) recovering the polypeptideexpressed by the cell; wherein the cell comprises a nucleic acid,wherein the nucleic acid encodes a polypeptide comprising the amino acidsequence of SEQ ID NO:
 8. 2. The method of claim 1, wherein the nucleicacid comprises the nucleotide sequence of SEQ ID NO:
 4. 3. The method ofclaim 1, wherein the nucleic acid comprises the nucleotide sequence ofSEQ ID NO:
 6. 4. The method of claim 3, wherein the mammalian cell is aCHO cell.
 5. The method of claim 1, wherein the amino terminus of thepolypeptide has the sequence ETR.
 6. The method of claim 1, wherein thehost cell is a mammalian cell.
 7. The method of claim 1, wherein thenucleic acid is operably linked to one or more regulatory sequences.